Anti-tryptase antibodies, compositions thereof, and uses thereof

ABSTRACT

The invention provides compositions including anti-tryptase antibodies and pharmaceutical compositions thereof, as well as methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/893,238, filed on Feb. 9, 2018, which claims benefit to U.S.Provisional Application No. 62/457,722, filed on Feb. 10, 2017, thecontents of which are incorporated herein by reference in their entirety

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 26, 2020, isnamed 50474-112004_Sequence_Listing_6.26.20_ST25 and is 108,979 bytes insize.

FIELD OF THE INVENTION

The invention relates to anti-tryptase antibodies, pharmaceuticalcompositions, and methods of using the same.

BACKGROUND

Human tryptase beta is a trypsin-like serine protease that is abundantin mast cells and, to a lesser extent, in basophils. Human tryptase beta(of which there are three subtypes, tryptase beta 1, tryptase beta 2,and tryptase beta 3) produced by the TPSAB1 and TPSB2 loci is thepredominant active tryptase produced by human mast cells. These two lociproduce four tryptase isoforms; TPSAB1 produces tryptase alpha andtryptase beta 1, while TPSB2 produces tryptase beta 2 and tryptase beta3. Tryptase alpha, as well as other isoforms such as tryptase gamma,tryptase delta, and tryptase epsilon are largely inactive.

The proteolytically processed, active tryptase beta is stored in thesecretory granules of mast cells as a tetramer in complex with heparin.Mast cell degranulation, which can be caused by IgE-dependent stimuli(e.g., allergens), or non-IgE-dependent stimuli (e.g., substance P oractive tryptase), leads to release of tryptase beta along with othergranule enzymes and histamine. Previous studies have observed increasedmast cell numbers in bronchial smooth muscle and epithelium of asthmapatients, as well as increased levels of tryptase beta in broncoalveolarlavage fluid. In addition, tryptase contributes to airwaybronchoconstriction and hyperresponsiveness, and has also been suggestedto play a role in fibrosis and extracellular matrix turnover, which arehallmarks of the airway remodeling process.

Tryptase has been suggested to be involved in various diseases anddisorders, including asthma and other pulmonary, inflammatory,autoimmune, and fibrotic disorders, for which there remains a need forimproved therapeutics, including therapeutic anti-tryptase antagonists,and methods of treatment. There have been attempts to develop smallmolecule tryptase inhibitors (see, e.g., Cairns, J. A., 2005, PulmonaryPharmacology & Therapeutics 18:55-66); however, to our knowledge, nobiologic tryptase antagonistic therapeutics, especially anti-tryptaseantagonistic antibodies, have been reported.

SUMMARY OF THE INVENTION

The present invention relates to anti-tryptase antibodies andpharmaceutical compositions thereof, as well as methods of using thesame.

In one aspect, the invention features an isolated antibody that binds tohuman tryptase beta 1, or an antigen-binding fragment thereof, whereinthe antibody comprises the following six hypervariable regions (HVRs):(a) an HVR-H1 comprising the amino acid sequence of DYGMV (SEQ ID NO:7); (b) an HVR-H2 comprising the amino acid sequence ofFISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).In some embodiments, the antibody is defined by the six HVRs comprisingthe amino acid sequence of SEQ ID NO: 7, 2, 8, 4, 5, and 6. In someembodiments, the antibody further comprises S43, P46, and W47 in thelight chain variable (VL) domain framework region L2 (FR-L2) (Kabatnumbering). In some embodiments, the antibody comprises (a) a heavychain variable (VH) domain comprising an amino sequence having at least90%, at least 95%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 9; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 90%, at least 95%, orat least 99% identity to the amino acid sequence of SEQ ID NO: 10; or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following VH domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 11); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 12);(c) an FR-H3 comprising the amino acid sequence ofRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR (SEQ ID NO: 13); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 9. In some embodiments, the antibody further comprises thefollowing VL domain FRs: (a) an FR-L1 comprising the amino acid sequenceof DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKSPKPWIY (SEQ ID NO: 16); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 17); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 18). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 10. In some embodiments, theantibody comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 76 and (b) a light chain comprising the amino acidsequence of SEQ ID NO: 77. In other embodiments, the antibody comprises(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 78and (b) a light chain comprising the amino acid sequence of SEQ ID NO:79.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises (a) a VH domain comprising an amino acidsequence having at least 90%, at least 95%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 9 and (b) a VL domaincomprising an amino acid sequence having at least 90%, at least 95%, orat least 99% sequence identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 9 and a VL domain comprising theamino acid sequence of SEQ ID NO: 10. In some embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 76 and (b) a light chain comprising the amino acid sequence of SEQID NO: 77. In other embodiments, the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 78 and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 79.

In another aspect, the invention features an isolated antibodycomprising (a) a heavy chain comprising the amino acid sequence of SEQID NO: 76 and (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 77.

In another aspect, the invention features an isolated antibodycomprising (a) a heavy chain comprising the amino acid sequence of SEQID NO: 78 and (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 79.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of DYGMV (SEQ ID NO: 7); (b) anHVR-H2 comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ IDNO: 2); (c) an HVR-H3 comprising the amino acid sequence of RDNYDWYFDV(SEQ ID NO: 29); (d) an HVR-L1 comprising the amino acid sequence ofSASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acidsequence of RTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising theamino acid sequence of QHYHSYPLT (SEQ ID NO: 6). In some embodiments,the antibody further comprises the following VH domain FRs: (a) an FR-H1comprising the amino acid sequence of EVKLVESGGGSVQPGGSRKLSCAASGFTFS(SEQ ID NO: 21); (b) an FR-H2 comprising the amino acid sequence ofWVRQAPGKGLEWVA (SEQ ID NO: 22); (c) an FR-H3 comprising the amino acidsequence of RFTISRDNPKNTLFLQMSSLRSEDTAMYYCAR (SEQ ID NO: 23); and (d) anFR-H4 comprising the amino acid sequence of WGTGTTVTVSS (SEQ ID NO: 24).In some embodiments, the VH domain comprises the amino acid sequence ofSEQ ID NO: 19. In some embodiments, the antibody further comprises thefollowing VL domain FRs: (a) an FR-L1 comprising the amino acid sequenceof QIVLTQSPAIMSASPGEKVTISC (SEQ ID NO: 25); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGSSPKPWIY (SEQ ID NO: 26); (c) an FR-L3comprising the amino acid sequence of GVPARFSGSGSGTSYSLTISSMEAEDAATYYC(SEQ ID NO: 27); and (d) an FR-L4 comprising the amino acid sequence ofFGAGTKLELK (SEQ ID NO: 28). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 20.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises (a) a VH domain comprising an aminosequence having at least 90%, at least 95% sequence, or at least 99%identity to the amino acid sequence of SEQ ID NO: 19; (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 20; or (c) a VH domainas in (a) and a VL domain as in (b). In some embodiments, the antibodyfurther comprises the following VH domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EVKLVESGGGSVQPGGSRKLSCAASGFTFS (SEQ ID NO:21); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 22); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNPKNTLFLQMSSLRSEDTAMYYCAR (SEQ ID NO: 23); and (d) an FR-H4comprising the amino acid sequence of WGTGTTVTVSS (SEQ ID NO: 24). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 19. In some embodiments, the antibody further comprises thefollowing VL domain FRs: (a) an FR-L1 comprising the amino acid sequenceof QIVLTQSPAIMSASPGEKVTISC (SEQ ID NO: 25); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGSSPKPWIY (SEQ ID NO: 26); (c) an FR-L3comprising the amino acid sequence of GVPARFSGSGSGTSYSLTISSMEAEDAATYYC(SEQ ID NO: 27); and (d) an FR-L4 comprising the amino acid sequence ofFGAGTKLELK (SEQ ID NO: 28). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 20.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,comprising (a) a VH domain comprising an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 19and (b) a VL domain comprising an amino acid sequence having at least99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

In some embodiments of any of the preceding aspects, the antibody bindsto an epitope on human tryptase beta 1 comprising at least one, at leasttwo, at least three, or all four residues selected from the groupconsisting of His51, Val80, Lys81, and Asp82 of SEQ ID NO: 71. In someembodiments, the antibody binds to an epitope on human tryptase beta 1comprising at least one, at least two, at least three, or all fourresidues selected from the group consisting of His51, Val80, Lys81, andAsp82 of SEQ ID NO: 71. In some embodiments, the antibody binds to anepitope on human tryptase beta 1 comprising His51 and at least one, atleast two, or all three residues selected from the group consisting ofVal80, Lys81, and Asp82 of SEQ ID NO: 71. In some embodiments, theepitope on human tryptase beta 1 further comprises one or more aminoacid residues selected from the group consisting of Gln67, Leu83, Ala84,Ala85, Arg87, Pro103, Val104, Ser105, Arg106, Glu128, Glu129, and Pro130of SEQ ID NO: 71. In some embodiments, the epitope on human tryptasebeta 1 comprises at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, or all twelve amino acid residues selectedfrom the group consisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103,Val104, Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71. Insome embodiments, the epitope on human tryptase beta 1 comprises His51,Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Va104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is determined by an X-raycrystallography model. In some embodiments, the antibody is capable ofdissociating both the small interface of tetrameric human tryptase beta1 and the large interface of tetrameric human tryptase beta 1.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of GYAIT (SEQ ID NO: 30); (b) anHVR-H2 comprising the amino acid sequence of GISSAATTFYSSWAKS (SEQ IDNO: 31); (c) an HVR-H3 comprising the amino acid sequence ofDPRGYGAALDRLDL (SEQ ID NO: 32); (d) an HVR-L1 comprising the amino acidsequence of QSIKSVYNNRLG (SEQ ID NO: 33); (e) an HVR-L2 comprising theamino acid sequence of ETSILTS (SEQ ID NO: 34); and (f) an HVR-L3comprising the amino acid sequence of AGGFDRSGDTT (SEQ ID NO: 35). Insome embodiments, the antibody is defined by the six HVRs comprising theamino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, and 35. In someembodiments, the antibody further comprises Arg71 and Va78 in VH domainFR-H3 (Kabat numbering). In some embodiments, the antibody furthercomprises the following VH domain FRs: (a) an FR-H1 comprising the aminoacid sequence of EVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO: 38); (b) anFR-H2 comprising the amino acid sequence of WIRQPPGKGLEWIG (SEQ ID NO:42); (c) an FR-H3 comprising the amino acid sequence ofRVTISRDTSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO: 43); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 41). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 36. In some embodiments, the antibody comprises the following VLdomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 64); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 65); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 66); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 63). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 37. In some embodiments, theantibody comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 80 and (b) a light chain comprising the amino acidsequence of SEQ ID NO: 81. In other embodiments, the antibody comprises(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 82and (b) a light chain comprising the amino acid sequence of SEQ ID NO:83.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises (a) a VH domain comprising an aminosequence having at least 90%, at least 95%, or at least 99% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs: 36, 47,48, 49, 50, 51, and 52; (b) a VL domain comprising an amino acidsequence having at least 90%, at least 95%, or at least 99% identity tothe amino acid sequence of any one of SEQ ID NO: 37, 53, 58, or 59; or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following VH domain FRs:(a) an FR-H1 comprising the amino acid sequence ofEVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO: 38); (b) an FR-H2 comprisingthe amino acid sequence of WIRQPPGKGLEWIG (SEQ ID NO: 42); (c) an FR-H3comprising the amino acid sequence of RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO: 43); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 41). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 36. In some embodiments,the antibody comprises the following VL domain FRs: (a) an FR-L1comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ IDNO: 64); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 65); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 66); and (d) anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 63).In some embodiments, the VL domain comprises the amino acid sequence ofSEQ ID NO: 37. In some embodiments, the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 80 and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 81. Inother embodiments, the antibody comprises (a) a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 82 and (b) a light chaincomprising the amino acid sequence of SEQ ID NO: 83.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase, or an antigen-binding fragment thereof, whereinthe antibody comprises (a) a VH domain comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 36 and (b) a VL domain comprisingan amino acid sequence having at least 90%, at least 95%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO: 37. Insome embodiments, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 36 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37. In some embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 80 and (b) a light chain comprising the amino acid sequence of SEQID NO: 81. In other embodiments, the antibody comprises (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 82 and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 83.

In another aspect, the invention features an isolated antibodycomprising (a) a heavy chain comprising the amino acid sequence of SEQID NO: 80 and (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 81.

In another aspect, the invention features an isolated antibodycomprising (a) a heavy chain comprising the amino acid sequence of SEQID NO: 82 and (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 83.

In another aspect, the invention features an isolated antibody thatbinds to human tryptase, or an antigen-binding fragment thereof, whereinthe antibody comprises (a) a VH domain comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 52 and (b) a VL domain comprisingan amino acid sequence having at least 90%, at least 95%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO: 53.

In some embodiments of any of the preceding aspects, the antibody bindsto an epitope on human tryptase beta 1 comprising at least one, at leasttwo, or all three residues selected from the group consisting of Gln100,Leu101, and Leu102 of SEQ ID NO: 71. In some embodiments, the epitope onhuman tryptase beta 1 further comprises one or more amino acid residuesselected from the group consisting of Trp55, Gln67, Asp82, Leu83, Ala84,Arg87, Pro103, Val104, Ser105, Arg106, Glu126, Leu127, Glu128, andGlu129 of SEQ ID NO: 71. In some embodiments, the epitope on humantryptase beta 1 comprises at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,or all fourteen amino acid residues selected from the group consistingof Trp55, Gln67, Asp82, Leu83, Ala84, Arg87, Pro103, Val104, Ser105,Arg106, Glu126, Leu127, Glu128, and Glu129 of SEQ ID NO: 71. In someembodiments, the epitope comprises Gln35, Trp55, Gln67, Asp82, Leu83,Ala84, Arg87, Gln100, Leu101, Leu102, Pro103, Val104, Ser105, Arg106,Glu126, Leu127, Glu128, Glu129, and Arg216 of SEQ ID NO: 71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is relative to a humantryptase beta 1 tetramer, and the epitope on human tryptase beta 1further comprises one or both of Gln35 and Arg216 of SEQ ID NO: 71. Insome embodiments, the epitope is determined by an X-ray crystallographymodel. In some embodiments, the antibody is capable of dissociating thesmall interface and/or the large interface of human tryptase beta 1.

In some embodiments of any of the preceding aspects, the antibodyfurther binds cynomolgus monkey (cyno) tryptase. In some embodiments,the antibody further binds human tryptase alpha. In some embodiments,the antibody further binds human tryptase beta 2 or human tryptase beta3. In some embodiments, the antibody binds human tryptase beta 2 andhuman tryptase beta 3.

In some embodiments of any of the preceding aspects, the antibody bindsthe tryptase with a K_(D) of about 1 nM or less. In some embodiments,the K_(D) is measured by a surface plasmon resonance (SPR) assay. Insome embodiments, the antibody binds the tryptase with a K_(D) ofbetween about 120 pM and about 0.5 nM. In some embodiments, the antibodybinds the tryptase with a K_(D) of between about 120 pM and about 300pM. In some embodiments, the antibody binds the tryptase with a K_(D) ofbetween about 120 pM and about 200 pM. In some embodiments, the antibodybinds the tryptase with a K_(D) of about 180 pM. In some embodiments,the antibody binds tryptase with a K_(D) of about 400 pM. In someembodiments, the SPR assay is performed at 25° C. In some embodiments,the K_(D) is measured using a BIACORE® SPR assay, for example, asdescribed in Example 1, Section (A)(vii). In some embodiments, the SPRassay can use a BIAcore® T200 or an equivalent device. In someembodiments, BIAcore® Series S CM5 sensor chips (or equivalent sensorchips) are immobilized with monoclonal mouse anti-human IgG (Fc)antibody and anti-tryptase antibodies are subsequently captured on theflow cell. Serial 3-fold dilutions of the His-tagged human tryptase beta1 monomer (SEQ ID NO: 128) are injected at a flow rate of 30 μl/min.Each sample is analyzed with 3 min association and 10 min dissociation.The assay is performed at 25° C. After each injection, the chip isregenerated using 3 M MgCl₂. Binding response is corrected bysubtracting the response units (RU) from a flow cell capturing anirrelevant IgG at similar density. A 1:1 Languir model of simultaneousfitting of k_(on) and k_(off) is used for kinetics analysis.

In some embodiments of any of the preceding aspects, the antibody iscapable of inhibiting the enzymatic activity of human tryptase beta 1.In some embodiments, the antibody inhibits the activity of tryptase withan IC50 of about 2.5 nM or lower as determined by a human tryptase betaenzymatic assay using a synthetic peptide S-2288 as a substrate. In someembodiments, the antibody inhibits the activity of tryptase with an IC50of between about 550 pM and about 2.5 nM. In some embodiments, theantibody inhibits the activity of tryptase with an IC50 of between about500 pM and about 2 nM. In some embodiments, the antibody inhibits theactivity of tryptase with an IC50 of between about 550 nM and 1.5 nM. Insome embodiments, the antibody inhibits the activity of tryptase with anIC50 of between about 500 pM and about 700 pM. In some embodiments, theinhibitory activity of the antibody is determined as described inExample 1(A)(viii)(a)). In some embodiments, the final concentration ofheparin in the human tryptase beta enzymatic assay using the syntheticpeptide S-2288 is 66 pg/ml. In some embodiments, recombinant humantryptase beta 1 tetramer active enzyme is diluted to 0.75 nM in TNHBuffer (200 mM Tris, 150 mM NaCl, 0.1 mg/mL heparin, 0.01% TRITON™X-100, pH 8.0), and combined 1:1 with anti-tryptase antibodies (dilutedin PBS) in 384-well plates. Plates are incubated for 1 h at ambienttemperature with gentle agitation. Colorimetric substrate S-2288(Chromogenix, Part No. 82-0852-39), or an equivalent substrate, isdiluted to 1200 μM in TNH Buffer and added to the plate. In someembodiments, the final in-well concentrations are 400 μM S-2288, 0.25 nMrecombinant human tryptase beta 1 tetramer, 66 pg/mL heparin, and from0.10 to 222 nM anti-tryptase antibody. Plates are incubated for 40 minat ambient temperature with gentle agitation and then read at A₄₀₅. TheIC50 of the anti-tryptase antibodies is determined from a four-parameterfit of their respective curves.

In some embodiments of any of the preceding aspects, the antibody iscapable of inhibiting the enzymatic activity of human tryptase beta 1 atpH 6. In particular, the inhibitory activity of the antibody may bedetermined at pH 6.

In some embodiments, the antibody is capable inhibitingtryptase-mediated stimulation of bronchial smooth muscle cellproliferation and/or collagen-based contraction. In some embodiments,the antibody is capable of inhibiting mast cell histamine release. Insome embodiments, the antibody is capable of inhibiting IgE-triggeredhistamine release and/or tryptase-triggered histamine release. In someembodiments, the antibody is capable of inhibiting cyno tryptase D1 asassessed by an active tryptase ELISA assay. In some embodiments, theantibody is capable of inhibiting tryptase activity in cynomolgus monkeybroncheoloar lavage (BAL) or nasosorption samples. In some embodiments,the antibody is capable of dissociating tetrameric human tryptasebeta 1. In some embodiments, the antibody is capable of dissociatingtetrameric human tryptase beta 1 when in a monovalent format. In someembodiments, the monovalent format is a Fab format. In some embodiments,the antibody is capable of dissociating tetrameric human tryptase beta 1in the presence of heparin. In some embodiments, the antibody is capableof dissociating tetrameric tryptase beta in the presence of 66 μg/mlheparin.

In another aspect, the invention features an antibody that binds to thesame epitope as any one of the preceding antibodies. In someembodiments, whether the antibody binds to the same epitope or competesfor binding to human tryptase beta 1 is determined by an epitope binningassay. In some embodiments, the epitope binning assay is an OCTET®epitope binning assay such as described in Example 3, Section C. In someembodiments, human tryptase beta 1 monomer protein is biotinylated atLys residue by reacting with NHS-PEG4-biotin. Biotinylated monomer isdiluted to 5 pg/ml in kinetics buffer (ForteBio, Inc.) and immobilizedonto streptavidin sensor tips (ForteBio, Inc.). After the immobilizationstep, human tryptase beta 1-immobilized sensors are saturated with thefirst antibody, diluted at 10-20 pg/ml, followed by binding with secondantibody diluted at 2.5 pg/ml. In some embodiments, the epitope binningassay is performed at 30° C.

In another aspect, the invention features an antibody that competes forbinding to human tryptase beta 1 with, or cross-blocks or iscross-blocked by any one of the preceding antibodies.

In some embodiments of any of the preceding aspects, the antibody ismonoclonal, human, humanized, or chimeric. In some embodiments, theantibody is humanized.

In some embodiments of any of the preceding aspects, the antibody is anantibody fragment that binds tryptase. In some embodiments, the antibodyfragment is selected from the group consisting of Fab, Fab′-SH, Fv,scFv, and (Fab′)₂ fragments.

In some embodiments of any of the preceding aspects, the antibody is afull-length antibody. In some embodiments, the antibody is an IgGantibody. In some embodiments, the IgG antibody is an IgG1 antibody. Insome embodiments, the IgG antibody is an IgG4 antibody. In someembodiments, the IgG4 antibody comprises a mutation in the hinge region.In some embodiments, the mutation is a substitution mutation. In someembodiments, the substitution mutation is at amino acid residue S228 (EUnumbering). In some embodiments, the IgG4 antibody comprises an S228Pmutation (EU numbering).

In some embodiments of any of the preceding aspects, the antibody is amonospecific antibody.

In some embodiments of any of the preceding aspects, the antibody is amultispecific antibody. In some embodiments, the antibody is abispecific antibody. In some embodiments, the antibody comprises a firstbinding domain that binds to tryptase and a second binding domain thatbinds to a second biological molecule, wherein the second biologicalmolecule is selected from the group consisting of interleukin-13(IL-13), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-17(IL-17), IgE, and interleukin-33 (IL-33). In some embodiments, thesecond biological molecule is IL-13. In some embodiments, the secondbiological molecule is IL-33. In some embodiments, the second biologicalmolecule is IgE.

In another aspect, the invention features an isolated nucleic acidencoding any of the antibodies described herein or a set of isolatednucleic acids together encoding the antibody.

In another aspect, the invention features an isolated nucleic acidencoding an antibody comprising a VH domain comprising the amino acidsequence of SEQ ID NO: 9 and/or a VL domain comprising the amino acidsequence of SEQ ID NO: 10, or a set of isolated nucleic acids togetherencoding the antibody, wherein the nucleic acid comprises a sequencethat is at least 85%, at least 90%, at least 95%, or at least 99%identical to the sequence of SEQ ID NO: 104 and/or SEQ ID NO: 105. Insome embodiments, the antibody comprises (a) a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 76 and/or (b) a light chaincomprising the amino acid sequence of SEQ ID NO: 77, and wherein thenucleic acid or set comprises a sequence that is at least 85%, at least90%, at least 95%, or at least 99% identical to the sequence of SEQ IDNO: 106 and/or SEQ ID NO: 107. In some embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 78 and/or (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 79, and wherein the nucleic acid or set comprises a sequencethat is at least 85%, at least 90%, at least 95%, or at least 99%identical to the sequence of SEQ ID NO: 108 and/or SEQ ID NO: 107. Insome embodiments, the nucleic acid or set comprises the sequence of SEQID NO: 108 and/or SEQ ID NO: 107.

In another aspect, the invention features an isolated nucleic acidencoding the antibody comprising a VH domain comprising the amino acidsequence of SEQ ID NO: 36 and/or a VL domain comprising the amino acidsequence of SEQ ID NO: 37, or a set of isolated nucleic acids togetherencoding the antibody, wherein the nucleic acid comprises a sequencethat is at least 85%, at least 90%, at least 95%, or at least 99%identical to the sequence of SEQ ID NO: 109 and/or SEQ ID NO: 110. Insome embodiments, the antibody comprises (a) a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 80 and/or (b) a light chaincomprising the amino acid sequence of SEQ ID NO: 81, and wherein thenucleic acid or set comprises a sequence that is at least 85%, at least90%, at least 95%, or at least 99% identical to the sequence of SEQ IDNO: 111 and/or SEQ ID NO: 112. In some embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 82 and/or (b) a light chain comprising the amino acid sequence ofSEQ ID NO: 83, and wherein the nucleic acid or set comprises a sequencethat is at least 85%, at least 90%, at least 95%, or at least 99%identical to the sequence of SEQ ID NO: 113 and/or SEQ ID NO: 112. Insome embodiments, the nucleic acid or set comprises the sequence of SEQID NO: 113 and/or SEQ ID NO: 112.

In another aspect, the invention features a vector (e.g., an expressionvector) or set of vectors comprising any of the isolated nucleic acidsor set of isolated nucleic acids described herein. In another aspect,the invention features host cells comprising the preceding nucleic acidsand/or vectors and/or sets of nucleic acids and/or sets of vectors. Insome embodiments, the host cell is a mammalian cell. In someembodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell.In some embodiments, the host cell is a prokaryotic cell. In someembodiments, the prokaryotic cell is E. coli.

In another aspect, the invention features a method of producing any ofthe antibodies described herein, the method comprising culturing a hostcell that comprises any of the preceding vectors (e.g., expressionvectors) or set of vectors in a culture medium under suitable conditionsthat allow production of the antibody. In some embodiments, the methodfurther comprises recovering the antibody from the host cell or theculture medium.

In another aspect, the invention features composition (e.g., apharmaceutical composition) comprising any one of the precedingantibodies. In some embodiments, the composition further comprises apharmaceutically acceptable carrier, excipient, or diluent.

In another aspect, the invention features a pharmaceutical compositioncomprising an isolated monoclonal antibody that binds to human tryptasebeta 1, or an antigen-binding fragment thereof, and a pharmaceuticallyacceptable carrier, excipient, or diluent, wherein the antibody binds tomonomeric tryptase beta 1 with a K_(D) of about 0.1 nM to about 1 nM,and/or wherein the antibody is capable of inhibiting the enzymaticactivity of the tryptase with a half-maximal inhibitory concentration(IC50) of about 0.1 nM to about 5 nM as determined by an in vitrotryptase enzymatic assay using S-2288 as a substrate.

In some embodiments of the preceding aspect, the antibody binds thetryptase with a K_(D) between about 0.5 nM to about 1 nM. In someembodiments, the antibody binds the tryptase with a K_(D) between about0.1 nM to about 0.5 nM. In some embodiments, the antibody binds thetryptase with a K_(D) of about 0.4 nM. In some embodiments, the antibodybinds the tryptase with a K_(D) of about 0.2 nM. In some embodiments,the K_(D) is measured by a surface plasmon resonance (SPR) assay. Insome embodiments, the SPR assay is performed at 25° C. In someembodiments, the K_(D) is measured using a BIACORE® SPR assay, forexample, as described in Example 1, Section (A)(vii). In someembodiments, the SPR assay can use a BIAcore® T200 or an equivalentdevice. In some embodiments, BIAcore® Series S CM5 sensor chips (orequivalent sensor chips) are immobilized with monoclonal mouseanti-human IgG (Fc) antibody and anti-tryptase antibodies aresubsequently captured on the flow cell. Serial 3-fold dilutions of theHis-tagged human tryptase beta 1 monomer (SEQ ID NO: 128) are injectedat a flow rate of 30 μl/min. Each sample is analyzed with 3 minassociation and 10 min dissociation. The assay is performed at 25° C.After each injection, the chip is regenerated using 3 M MgCl₂. Bindingresponse is corrected by subtracting the response units (RU) from a flowcell capturing an irrelevant IgG at similar density. A 1:1 Languir modelof simultaneous fitting of k_(on) and k_(off) is used for kineticsanalysis.

In some embodiments of the preceding aspect, the antibody is capable ofinhibiting the activity of the tryptase with an IC50 of about 0.5 nM toabout 5 nM. In some embodiments, the antibody is capable of inhibitingthe activity of the tryptase with an IC50 of about 0.1 nM to about 2 nM.In some embodiments, the antibody is capable of inhibiting the activityof human tryptase with an IC50 of about 4 nM. In some embodiments, theantibody is capable of inhibiting the activity of the tryptase with anIC50 of about 0.6 nM. In some embodiments, the antibody is capable ofinhibiting the tryptase activity at pH 6 in an in vitro tryptaseenzymatic assay using S-2288 as a substrate. In some embodiments, theinhibitory activity of the antibody is determined as described in theExamples (e.g., Example 1, Section (A)(viii)(a)). In some embodiments,recombinant human tryptase beta 1 tetramer active enzyme is diluted to0.75 nM in TNH Buffer (200 mM Tris, 150 mM NaCl, 0.1 mg/mL heparin,0.01% TRITON™ X-100, pH 8.0), and combined 1:1 with anti-tryptaseantibodies (diluted in PBS) in 384-well plates. Plates are incubated for1 h at ambient temperature with gentle agitation. Colorimetric substrateS-2288 (Chromogenix, Part No. 82-0852-39), or an equivalent substrate,is diluted to 1200 pM in TNH Buffer and added to the plate. In someembodiments, the final in-well concentrations are 400 pM S-2288, 0.25 nMrecombinant human tryptase beta 1 tetramer, 66 pg/mL heparin, and from0.10 to 222 nM anti-tryptase antibody. Plates are incubated for 40 minat ambient temperature with gentle agitation and then read at A₄₀₅. TheIC50 of the anti-tryptase antibodies is determined from a four-parameterfit of their respective curves. In some embodiments, the finalconcentration of heparin in the human tryptase beta enzymatic assayusing the synthetic peptide S-2288 is 66 pg/ml.

In some embodiments of the preceding aspect, the antibody is capableinhibiting tryptase-mediated stimulation of bronchial smooth muscle cellproliferation and/or collagen-based contraction. In some embodiments,the antibody is capable of inhibiting mast cell histamine release. Insome embodiments, the antibody is capable of inhibiting IgE-triggeredhistamine release and/or tryptase-triggered histamine release. In someembodiments, the antibody is capable of inhibiting tryptase activity incynomolgus monkey broncheoloar lavage (BAL) or nasosorption samples. Insome embodiments, the antibody is capable of dissociating tetramerichuman tryptase beta 1. In some embodiments, the antibody is capable ofdissociating tetrameric human tryptase beta 1 when in a monovalentformat. In some embodiments, the monovalent format is a Fab format. Insome embodiments, the antibody is capable of dissociating tetramerichuman tryptase beta 1 in the presence of heparin. In some embodiments,the antibody is capable of dissociating tetrameric tryptase beta in thepresence of 66 pg/ml heparin.

In some embodiments of the preceding aspect, the antibody comprises thefollowing six hypervariable regions (HVRs): (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6). In some embodiments, the antibody comprises(a) a heavy chain variable (VH) domain comprising an amino sequencehaving at least 90%, at least 95%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 9; (b) a light chain variable (VL)domain comprising an amino acid sequence having at least 90%, at least95%, or at least 99% identity to the amino acid sequence of SEQ ID NO:10; or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following VH domain FRs:(a) an FR-H1 comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 11); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 12); (c) an FR-H3comprising the amino acid sequence of RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR(SEQ ID NO: 13); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 14). In some embodiments, the VH domain of theantibody comprises the amino acid sequence of SEQ ID NO: 9. In someembodiments, the antibody further comprises the following VL domain FRs:(a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKSPKPWIY (SEQ ID NO: 16); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 17); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 18). In some embodiments, the VL domain of theantibody comprises the amino acid sequence of SEQ ID NO: 10. In someembodiments, the antibody comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 76 and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 77. In some embodiments, theantibody comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 78 and (b) a light chain comprising the amino acidsequence of SEQ ID NO: 79. In some embodiments, the antibody binds to anepitope on human tryptase beta 1 comprising at least one, at least two,at least three, or all four residues selected from the group consistingof His51, Val80, Lys81, and Asp82 of SEQ ID NO: 71. In some embodiments,the antibody binds to an epitope on human tryptase beta 1 comprisingHis51 and at least one, at least two, or all three residues selectedfrom the group consisting of Val 80, Lys81, and Asp82 of SEQ ID NO: 71.In some embodiments, the epitope on human tryptase beta 1 furthercomprises one or more amino acid residues selected from the groupconsisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103, Va104, Ser105,Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71. In someembodiments, the epitope on human tryptase beta 1 comprises at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, at leasteleven, or all twelve amino acid residues selected from the groupconsisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103, Val104, Ser105,Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71. In someembodiments, the epitope on human tryptase beta 1 comprises His51,Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Val104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is determined by an X-raycrystallography model. In some embodiments, the antibody is capable ofdissociating both the small interface of tetrameric human tryptase beta1 and the large interface of tetrameric human tryptase beta 1.

In other embodiments of the preceding aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofGYAIT (SEQ ID NO: 30); (b) an HVR-H2 comprising the amino acid sequenceof GISSAATTFYSSWAKS (SEQ ID NO: 31); (c) an HVR-H3 comprising the aminoacid sequence of DPRGYGAALDRLDL (SEQ ID NO: 32); (d) an HVR-L1comprising the amino acid sequence of QSIKSVYNNRLG (SEQ ID NO: 33); (e)an HVR-L2 comprising the amino acid sequence of ETSILTS (SEQ ID NO: 34);and (f) an HVR-L3 comprising the amino acid sequence of AGGFDRSGDTT (SEQID NO: 35). In some embodiments, the antibody comprises (a) a VH domaincomprising an amino sequence having at least 90%, at least 95%, or atleast 99% sequence identity to the amino acid sequence of any one of SEQID NOs: 36, 47, 48, 49, 50, 51, and 52; (b) a VL domain comprising anamino acid sequence having at least 90%, at least 95%, or at least 99%identity to the amino acid sequence of any one of SEQ ID NO: 37, 53, 58,or 59; or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following VH domain FRs:(a) an FR-H1 comprising the amino acid sequence ofEVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO: 38); (b) an FR-H2 comprisingthe amino acid sequence of WIRQPPGKGLEWIG (SEQ ID NO: 42); (c) an FR-H3comprising the amino acid sequence of RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO: 43); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 41). In some embodiments, the VH domain of theantibody comprises the amino acid sequence of SEQ ID NO: 36. In someembodiments, the antibody further comprises the following VL domain FRs:(a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 64); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 65); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 66); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 63). In some embodiments, the VL domain of theantibody comprises the amino acid sequence of SEQ ID NO: 37. In someembodiments, the antibody comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 80 and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 81. In some embodiments, theantibody comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 82 and (b) a light chain comprising the amino acidsequence of SEQ ID NO: 83. In some embodiments, the antibody binds to anepitope on human tryptase beta 1 comprising at least one, at least two,or all three residues selected from the group consisting of Gln100,Leu101, and Leu102 of SEQ ID NO: 71. In some embodiments, the epitope onhuman tryptase beta 1 further comprises one or more amino acid residuesselected from the group consisting of Trp55, Gln67, Asp82, Leu83, Ala84,Arg87, Pro103, Val104, Ser105, Arg106, Glu126, Leu127, Glu128, andGlu129 of SEQ ID NO: 71. In some embodiments, the epitope on humantryptase beta 1 comprises at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,or all fourteen amino acid residues selected from the group consistingof Trp55, Gln67, Asp82, Leu83, Ala84, Arg87, Pro103, Val104, Ser105,Arg106, Glu126, Leu127, Glu128, and Glu129 of SEQ ID NO: 71. In someembodiments, the epitope comprises Gln35, Trp55, Gln67, Asp82, Leu83,Ala84, Arg87, Gln100, Leu101, Leu102, Pro103, Va104, Ser105, Arg106,Glu126, Leu127, Glu128, Glu129, and Arg216 of SEQ ID NO: 71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is relative to a humantryptase beta 1 tetramer, and the epitope on human tryptase beta 1further comprises one or both of Gln35 and Arg216 of SEQ ID NO: 71. Insome embodiments, the epitope is determined by an X-ray crystallographymodel. In some embodiments, the antibody is capable of dissociating thesmall interface and/or the large interface of human tryptase beta 1.

In another aspect, the invention features a composition (e.g., apharmaceutical composition) comprising an isolated monoclonal antibodythat binds to human tryptase beta 1, or an antigen-binding fragmentthereof, and a pharmaceutically acceptable carrier, excipient, ordiluent, wherein the antibody binds to an epitope on human tryptase beta1 comprising at least one, at least two, at least three, or all fourresidues selected from the group consisting of His51, Val80, Lys81, andAsp82 of SEQ ID NO: 71. In some embodiments, the antibody binds to anepitope on human tryptase beta 1 comprising His51 and at least one, atleast two, or all three residues selected from the group consisting ofVal80, Lys81, and Asp82 of SEQ ID NO: 71. In some embodiments, theepitope on human tryptase beta 1 further comprises one or more aminoacid residues selected from the group consisting of Gln67, Leu83, Ala84,Ala85, Arg87, Pro103, Val104, Ser105, Arg106, Glu128, Glu129, and Pro130of SEQ ID NO: 71. In some embodiments, the epitope on human tryptasebeta 1 comprises at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, or all twelve amino acid residues selectedfrom the group consisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103,Val104, Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71. Insome embodiments, the epitope on human tryptase beta 1 comprises His51,Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Va104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is determined by an X-raycrystallography model. In some embodiments, the antibody is capable ofdissociating both the small interface of tetrameric human tryptase beta1 and the large interface of tetrameric human tryptase beta 1. In someembodiments, the antibody comprises the following six hypervariableregions (HVRs): (a) an HVR-H1 comprising the amino acid sequence ofDYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprising the amino acid sequenceof FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).In some embodiments, the antibody comprises (a) a heavy chain variable(VH) domain comprising an amino sequence having at least 90%, at least95%, or at least 99% sequence identity to the amino acid sequence of SEQID NO: 9; (b) a light chain variable (VL) domain comprising an aminoacid sequence having at least 90%, at least 95%, or at least 99%identity to the amino acid sequence of SEQ ID NO: 10; or (c) a VH domainas in (a) and a VL domain as in (b). In some embodiments, the antibodyfurther comprises the following VH domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:11); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 12); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR (SEQ ID NO: 13); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). Insome embodiments, the VH domain of the antibody comprises the amino acidsequence of SEQ ID NO: 9. In some embodiments, the antibody furthercomprises the following VL domain FRs: (a) an FR-L1 comprising the aminoacid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKSPKPWIY (SEQ ID NO: 16);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 18). Insome embodiments, the VL domain of the antibody comprises the amino acidsequence of SEQ ID NO: 10. In some embodiments, the antibody comprises(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 76and (b) a light chain comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, the antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 78 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO: 79.

In another aspect, the invention features a composition (e.g., apharmaceutical composition) comprising an isolated monoclonal antibodythat binds to human tryptase beta 1, or an antigen-binding fragmentthereof, and a pharmaceutically acceptable carrier, excipient, ordiluent, wherein the antibody binds to an epitope on human tryptase beta1 comprising at least one, at least two, or all three residues selectedfrom the group consisting of Gln100, Leu101, and Leu102 of SEQ ID NO:71. In some embodiments, the epitope on human tryptase beta 1 furthercomprises one or more amino acid residues selected from the groupconsisting of Trp55, Gln67, Asp82, Leu83, Ala84, Arg87, Pro103, Val104,Ser105, Arg106, Glu126, Leu127, Glu128, and Glu129 of SEQ ID NO: 71. Insome embodiments, the epitope on human tryptase beta 1 comprises atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, at leasteleven, at least twelve, at least thirteen, or all fourteen amino acidresidues selected from the group consisting of Trp55, Gln67, Asp82,Leu83, Ala84, Arg87, Pro103, Val104, Ser105, Arg106, Glu126, Leu127,Glu128, and Glu129 of SEQ ID NO: 71. In some embodiments, the epitopecomprises Gln35, Trp55, Gln67, Asp82, Leu83, Ala84, Arg87, Gln100,Leu101, Leu102, Pro103, Val104, Ser105, Arg106, Glu126, Leu127, Glu128,Glu129, and Arg216 of SEQ ID NO: 71. In some embodiments, the epitope isrelative to a human tryptase beta 1 monomer or tetramer. In someembodiments, the epitope is relative to a human tryptase beta 1tetramer, and the epitope on human tryptase beta 1 further comprises oneor both of Gln35 and Arg216 of SEQ ID NO: 71. In some embodiments, theepitope is determined by an X-ray crystallography model. In someembodiments, the antibody is capable of dissociating the small interfaceand/or the large interface of human tryptase beta 1. In someembodiments, the antibody comprises the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of GYAIT (SEQ ID NO: 30); (b)an HVR-H2 comprising the amino acid sequence of GISSAATTFYSSWAKS (SEQ IDNO: 31); (c) an HVR-H3 comprising the amino acid sequence ofDPRGYGAALDRLDL (SEQ ID NO: 32); (d) an HVR-L1 comprising the amino acidsequence of QSIKSVYNNRLG (SEQ ID NO: 33); (e) an HVR-L2 comprising theamino acid sequence of ETSILTS (SEQ ID NO: 34); and (f) an HVR-L3comprising the amino acid sequence of AGGFDRSGDTT (SEQ ID NO: 35). Insome embodiments, the antibody comprises (a) a VH domain comprising anamino sequence having at least 90%, at least 95%, or at least 99%sequence identity to the amino acid sequence of any one of SEQ ID NOs:36, 47, 48, 49, 50, 51, and 52; (b) a VL domain comprising an amino acidsequence having at least 90%, at least 95%, or at least 99% identity tothe amino acid sequence of any one of SEQ ID NO: 37, 53, 58, or 59; or(c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following VH domain FRs:(a) an FR-H1 comprising the amino acid sequence ofEVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO: 38); (b) an FR-H2 comprisingthe amino acid sequence of WIRQPPGKGLEWIG (SEQ ID NO: 42); (c) an FR-H3comprising the amino acid sequence of RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO: 43); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 41). In some embodiments, the VH domain of theantibody comprises the amino acid sequence of SEQ ID NO: 36. In someembodiments, the antibody further comprises the following VL domain FRs:(a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 64); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 65); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 66); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 63). In some embodiments, the VL domain of theantibody comprises the amino acid sequence of SEQ ID NO: 37. In someembodiments, the antibody comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 80 and (b) a light chain comprisingthe amino acid sequence of SEQ ID NO: 81. In some embodiments, theantibody comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 82 and (b) a light chain comprising the amino acidsequence of SEQ ID NO: 83.

In some embodiments, the antibody is capable of further binding to humantryptase alpha, tryptase beta 2, tryptase beta 3 and/or cyno tryptaseD1.

In any of the preceding compositions (e.g., pharmaceuticalcompositions), the antibody may be monoclonal, human, humanized, orchimeric. In some embodiments, the antibody is humanized.

In any of the preceding compositions (e.g., pharmaceuticalcompositions), the composition may be for use in a human.

Any of the preceding compositions (e.g., pharmaceutical compositions)may be lyophilized. In other embodiments, any of the precedingcompositions (e.g., pharmaceutical compositions) may be a liquid.

In any of the preceding compositions (e.g., pharmaceuticalcompositions), the excipient may be an antioxidant. In some embodiments,the composition comprises one or more antioxidants selected from thegroup consisting of N-acetyltryptophan, tryptophan, methionine,cysteine, glutathione, thiosorbitol, ascorbic acid, monothioglycerol,cyclodextrins, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid), pyridoxine, mannitol, and a metal chelator. In some embodiments,the composition comprises N-acetyltryptophan or methionine. In someembodiments, the composition comprises N-acetyltryptophan andmethionine.

In another aspect, the invention features a composition (e.g., apharmaceutical composition) comprising: (i) an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises the following six hypervariable regions(HVRs): (a) an HVR-H1 comprising the amino acid sequence of DYGMV (SEQID NO: 7); (b) an HVR-H2 comprising the amino acid sequence ofFISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6),wherein the oxidation of tryptophan at position 6 of HVR-H3 (SEQ ID NO:8) is no more than 30%. In some embodiments, the oxidation of tryptophanat position 6 of HVR-H3 (SEQ ID NO: 8) is no more than 28%, 25%, 20%,15%, 10%, or 6%. In some embodiments, the oxidation of tryptophan atposition 6 of HVR-H3 (SEQ ID NO: 8) is determined following an AAPHstress test. In some embodiments, the oxidation of tryptophan atposition 6 of HVR-H3 (SEQ ID NO: 8) is determined within one year fromthe initial production of the composition.

Any of the preceding compositions (e.g., pharmaceutical compositions)may comprise N-acetyltryptophan at a concentration of about 0.1 mM toabout 5 mM. In some embodiments, the concentration of N-acetyltryptophanis about 0.1 mM to about 1 mM. In some embodiments, the concentration ofN-acetyltryptophan is about 0.3 mM. In some embodiments, the compositioncomprises methionine at a concentration of about 1 mM to about 20 mM. Insome embodiments, the concentration of methionine is about 1 mM to about10 mM. In some embodiments, the concentration of methionine is about 5mM.

In another aspect, the invention features a composition (e.g., apharmaceutical composition) comprising: (i) an isolated antibody thatbinds to human tryptase, or an antigen-binding fragment thereof, whereinthe antibody comprises the following six hypervariable regions (HVRs):(a) an HVR-H1 comprising the amino acid sequence of DYGMV (SEQ ID NO:7); (b) an HVR-H2 comprising the amino acid sequence ofFISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6);(ii) N-acetyltryptophan at a concentration of about 0.1 mm to about 1mM; and (iii) methionine at a concentration of about 1 mM to about 10mM.

In any of the preceding compositions (e.g., pharmaceuticalcompositions), the antibody concentration may be about 1 mg/ml to about250 mg/ml. In some embodiments, the antibody concentration is about 150mg/ml.

Any of the preceding compositions (e.g., pharmaceutical compositions)may further include one or more additional excipients selected from thegroup consisting of a stabilizer, a buffer, a surfactant, and a tonicityagent. In some embodiments, the composition further comprises a buffer.In some embodiments, the buffer is arginine succinate and/or histidinesuccinate. In some embodiments, the buffer comprises arginine succinateand histidine succinate. In some embodiments, the concentration ofarginine succinate is about 50 mM to about 500 mM. In some embodiments,the concentration of arginine succinate is about 100 mM to about 300 mM.In some embodiments, the concentration of arginine succinate is about200 mM. In some embodiments, the concentration of histidine succinate isabout 1 mM to about 50 mM. In some embodiments, the concentration ofhistidine succinate is about 15 mM to about 25 mM. In some embodiments,the concentration of histidine succinate is about 20 mM. In someembodiments, the composition further comprises a surfactant. In someembodiments, the surfactant is poloxamer 188 or polysorbate 20. In someembodiments, the surfactant is poloxamer 188. In some embodiments, theconcentration of poloxamer 188 is about 0.005% to about 0.1%. In someembodiments, the concentration of poloxamer 188 is about 0.005% to about0.05%. In some embodiments, the concentration of poloxamer 188 is about0.02%. In some embodiments, the pH of the composition is about 4.5 toabout 7.0. In some embodiments, the pH of the composition is about 4.5to about 6.5. In some embodiments, the pH of the composition is about5.5. In some embodiments, the composition is in a light-proof container.In some embodiments, the composition is in a pre-filled syringe.

Any of the preceding compositions (e.g., pharmaceutical compositions)may further include an IL-13 axis binding antagonist, an IL-5 axisbinding antagonist, an IL-33 axis binding antagonist, an M1 primeantagonist, an IgE antagonist, a TRPA1 antagonist, a CRTH2 antagonist, abroncodilator or asthma symptom controller medication, animmunomodulator, a corticosteroid, a Th2 pathway inhibitor, a tyrosinekinase inhibitor, or a phosphodiesterase inhibitor. In some embodiments,the IL-13 axis binding antagonist is an anti-IL-13 antibody. In someembodiments, the anti-IL-13 antibody is lebrikizumab. In someembodiments, the IL-5 axis binding antagonist is an IL-5 bindingantagonist or an IL-5 receptor binding antagonist. In some embodiments,the IL-33 axis binding antagonist is an IL-33 binding antagonist or anST2 binding antagonist. In some embodiments, the IL-33 bindingantagonist is an anti-IL-33 antibody. In some embodiments, the M1 primeantagonist is quilizumab. In some instances, the IgE antagonist isomalizumab (XOLAIR®).

Any of the preceding compositions (e.g., pharmaceutical compositions)may be formulated for administration to a human. In certain embodiments,the pharmaceutical compositions comprise antibodies that do not comprisenon-human constant region sequences. In certain embodiments, thepharmaceutical compositions comprise antibodies that do not comprisenon-human framework and non-human constant region sequences. In certainembodiments, the pharmaceutical compositions comprise antibodies thatare human antibodies, humanized antibodies, or chimeric antibodies.

In some aspects, any one of the preceding antibodies can be used as amedicament.

In some aspects, any of the preceding antibodies can be used in treatinga disorder. In some embodiments, the disorder is selected from the groupconsisting of a pulmonary disorder, an autoimmune disorder, aninflammatory disorder, a fibrotic disorder, a granulocytic (neutrophilicor eosinophilic) disorder, a monocytic disorder, a lymphocytic disorder,or a disorder associated with increased numbers or distribution ofnormal or aberrant tissue resident cells (such as mast cells,macrophages, or lymphocytes) or stromal cells (such as fibroblasts,myofibroblasts, smooth muscle cells, epithelia, or endothelia). In someembodiments, the disorder is a pulmonary disorder. In some embodiments,the pulmonary disorder is selected from the group consisting of asthma,airway hyperresponsiveness, and chronic obstructive pulmonary disease(COPD). In some embodiments, the pulmonary disorder is asthma. In someembodiments, the asthma is Th2-high asthma or Th2-low asthma. In someembodiments, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, psoriasis, eosinophilic esophagitis,inflammatory bowel disease (IBD), and Crohn's disease. In someembodiments, the inflammatory disorder is chronic idiopathic urticaria(CIU, also known as chronic spontaneous urticaria, CSU), anaphylaxis,anaphylactic shock, atopic dermatitis, or allergic rhinitis. In someembodiments, the fibrotic disorder is idiopathic pulmonary fibrosis(IPF). In some embodiments, the disorder is a disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is mastocytosis. In someembodiments, the antibody is for use in combination with an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an IL-13 axis binding antagonist, an IL-5 axis binding antagonist, anIL-33 axis binding antagonist, an M1 prime antagonist, an IgEantagonist, a TRPA1 antagonist, a CRTH2 antagonist, a broncodilator orasthma symptom controller medication, an immunomodulator, acorticosteroid, a Th2 pathway inhibitor, a tyrosine kinase inhibitor, ora phosphodiesterase inhibitor. In some embodiments, the IL-13 axisbinding antagonist is an anti-IL-13 antibody. In some embodiments, theanti-IL-13 antibody is lebrikizumab. In some embodiments, the IL-5 axisbinding antagonist is an IL-5 binding antagonist or an IL-5 receptorbinding antagonist. In some embodiments, the IL-33 axis bindingantagonist is an IL-33 binding antagonist or an ST2 binding antagonist.In some embodiments, the IL-33 binding antagonist is an anti-IL-33antibody. In some embodiments, the M1 prime antagonist is quilizumab. Insome embodiments, the antibody is for administration subcutaneously,intravenously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the antibody is for administration subcutaneously. In some embodiments,the antibody is for use in a human subject.

In some aspects, any one of the preceding compositions (e.g.,pharmaceutical compositions) can be used as a medicament.

In some aspects, any of the preceding compositions (e.g., pharmaceuticalcompositions) can be used in treating a disorder. In some embodiments,the disorder is selected from the group consisting of a pulmonarydisorder, an autoimmune disorder, an inflammatory disorder, a fibroticdisorder, a granulocytic (neutrophilic or eosinophilic) disorder, amonocytic disorder, a lymphocytic disorder, or a disorder associatedwith increased numbers or distribution of normal or aberrant tissueresident cells (such as mast cells, macrophages, or lymphocytes) orstromal cells (such as fibroblasts, myofibroblasts, smooth muscle cells,epithelia, or endothelia). In some embodiments, the disorder is apulmonary disorder. In some embodiments, the pulmonary disorder isselected from the group consisting of asthma, airwayhyperresponsiveness, and chronic obstructive pulmonary disease (COPD).In some embodiments, the pulmonary disorder is asthma. In someembodiments, the asthma is Th2-high asthma or Th2-low asthma. In someembodiments, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, psoriasis, eosinophilic esophagitis,inflammatory bowel disease (IBD), and Crohn's disease. In someembodiments, the inflammatory disorder is chronic idiopathic urticaria(CIU or CSU), anaphylaxis, anaphylactic shock, atopic dermatitis, orallergic rhinitis. In some embodiments, the fibrotic disorder isidiopathic pulmonary fibrosis (IPF). In some embodiments, the disorderis a disorder associated with increased numbers or distribution ofnormal or aberrant tissue resident cells (such as mast cells,macrophages, or lymphocytes) or stromal cells (such as fibroblasts,myofibroblasts, smooth muscle cells, epithelia, or endothelia). In someembodiments, the disorder is mastocytosis. In some embodiments, thecomposition (e.g., pharmaceutical composition) is for use in combinationwith an additional therapeutic agent. In some embodiments, theadditional therapeutic agent is an IL-13 axis binding antagonist, anIL-5 axis binding antagonist, an IL-33 axis binding antagonist, an M1prime antagonist, an IgE antagonist, a TRPA1 antagonist, a CRTH2antagonist, a broncodilator or asthma symptom controller medication, animmunomodulator, a corticosteroid, a Th2 pathway inhibitor, a tyrosinekinase inhibitor, or a phosphodiesterase inhibitor. In some embodiments,the IL-13 axis binding antagonist is an anti-IL-13 antibody. In someembodiments, the anti-IL-13 antibody is lebrikizumab. In someembodiments, the IL-5 axis binding antagonist is an IL-5 bindingantagonist or an IL-5 receptor binding antagonist. In some embodiments,the IL-33 axis binding antagonist is an IL-33 binding antagonist or anST2 binding antagonist. In some embodiments, the IL-33 bindingantagonist is an anti-IL-33 antibody. In some embodiments, the M1 primeantagonist is quilizumab. In some embodiments, the composition (e.g.,pharmaceutical composition) is for administration subcutaneously,intravenously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the composition (e.g., pharmaceutical composition) is for administrationsubcutaneously. In some embodiments, the composition (e.g.,pharmaceutical composition) is for use in a human subject.

In some aspects, any one of the preceding antibodies can be used in themanufacture of a medicament for treating a disorder. In someembodiments, the disorder is selected from the group consisting of apulmonary disorder, an autoimmune disorder, an inflammatory disorder, afibrotic disorder, a granulocytic (neutrophilic or eosinophilic)disorder, a monocytic disorder, a lymphocytic disorder, or a disorderassociated with increased numbers or distribution of normal or aberranttissue resident cells (such as mast cells, macrophages, or lymphocytes)or stromal cells (such as fibroblasts, myofibroblasts, smooth musclecells, epithelia, or endothelia). In some embodiments, the disorder is apulmonary disorder. In some embodiments, the pulmonary disorder isselected from the group consisting of asthma, airwayhyperresponsiveness, and chronic obstructive pulmonary disease (COPD).In some embodiments, the pulmonary disorder is asthma. In someembodiments, the asthma is Th2-high asthma or Th2-low asthma. In someembodiments, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, psoriasis, eosinophilic esophagitis,inflammatory bowel disease (IBD), and Crohn's disease. In someembodiments, the inflammatory disorder is chronic idiopathic urticaria(CIU or CSU), anaphylaxis, anaphylactic shock, atopic dermatitis, orallergic rhinitis. In some embodiments, the fibrotic disorder isidiopathic pulmonary fibrosis (IPF). In some embodiments, the disorderis a disorder associated with increased numbers or distribution ofnormal or aberrant tissue resident cells (such as mast cells,macrophages, or lymphocytes) or stromal cells (such as fibroblasts,myofibroblasts, smooth muscle cells, epithelia, or endothelia). In someembodiments, the disorder is mastocytosis. In some embodiments, themedicament is formulated for use in combination with an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an IL-13 axis binding antagonist, an IL-5 axis binding antagonist, anIL-33 axis binding antagonist, an M1 prime antagonist, an IgEantagonist, a TRPA1 antagonist, a CRTH2 antagonist, a broncodilator orasthma symptom controller medication, an immunomodulator, acorticosteroid, a Th2 pathway inhibitor, a tyrosine kinase inhibitor, ora phosphodiesterase inhibitor. In some embodiments, the IL-13 axisbinding antagonist is an anti-IL-13 antibody. In some embodiments, theanti-IL-13 antibody is lebrikizumab. In some embodiments, the IL-5 axisbinding antagonist is an IL-5 binding antagonist or an IL-5 receptorbinding antagonist. In some embodiments, the IL-33 axis bindingantagonist is an IL-33 binding antagonist or an ST2 binding antagonist.In some embodiments, the IL-33 binding antagonist is an anti-IL-33antibody. In some embodiments, the M1 prime antagonist is quilizumab. Insome embodiments, the medicament is formulated for administrationsubcutaneously, intravenously, intramuscularly, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. In someembodiments, the medicament is formulated for administrationsubcutaneously. In some embodiments, the medicament is formulated foruse in a human subject.

In some aspects, any one of the preceding compositions (e.g.,pharmaceutical compositions) can be used in the manufacture of amedicament for treating a disorder. In some embodiments, the disorder isselected from the group consisting of a pulmonary disorder, anautoimmune disorder, an inflammatory disorder, a fibrotic disorder, agranulocytic (neutrophilic or eosinophilic) disorder, a monocyticdisorder, a lymphocytic disorder, or a disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is a pulmonary disorder.In some embodiments, the pulmonary disorder is selected from the groupconsisting of asthma, airway hyperresponsiveness, and chronicobstructive pulmonary disease (COPD). In some embodiments, the pulmonarydisorder is asthma. In some embodiments, the asthma is Th2-high asthmaor Th2-low asthma. In some embodiments, the autoimmune disorder isselected from the group consisting of rheumatoid arthritis, psoriasis,eosinophilic esophagitis, inflammatory bowel disease (IBD), and Crohn'sdisease. In some embodiments, the inflammatory disorder is chronicidiopathic urticaria (CIU or CSU), anaphylaxis, anaphylactic shock,atopic dermatitis, or allergic rhinitis. In some embodiments, thefibrotic disorder is idiopathic pulmonary fibrosis (IPF). In someembodiments the disorder is a disorder associated with increased numbersor distribution of normal or aberrant tissue resident cells (such asmast cells, macrophages, or lymphocytes) or stromal cells (such asfibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is mastocytosis. In someembodiments, the medicament is formulated for use in combination with anadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is an IL-13 axis binding antagonist, an IL-5 axisbinding antagonist, an IL-33 axis binding antagonist, an M1 primeantagonist, an IgE antagonist, a TRPA1 antagonist, a CRTH2 antagonist, abroncodilator or asthma symptom controller medication, animmunomodulator, a corticosteroid, a Th2 pathway inhibitor, a tyrosinekinase inhibitor, or a phosphodiesterase inhibitor. In some embodiments,the IL-13 axis binding antagonist is an anti-IL-13 antibody. In someembodiments, the anti-IL-13 antibody is lebrikizumab. In someembodiments, the IL-5 axis binding antagonist is an IL-5 bindingantagonist or an IL-5 receptor binding antagonist. In some embodiments,the IL-33 axis binding antagonist is an IL-33 binding antagonist or anST2 binding antagonist. In some embodiments, the IL-33 bindingantagonist is an anti-IL-33 antibody. In some embodiments, the M1 primeantagonist is quilizumab. In some embodiments, the medicament isformulated for administration subcutaneously, intravenously,intramuscularly, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. In some embodiments, the medicamentis formulated for administration subcutaneously. In some embodiments,the medicament is formulated for use in a human subject.

In another aspect, the invention features a method of treating adisorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyone of the preceding antibodies. In some embodiments, the disorder isselected from the group consisting of a pulmonary disorder, anautoimmune disorder, an inflammatory disorder, a fibrotic disorder, agranulocytic (neutrophilic or eosinophilic) disorder, a monocyticdisorder, a lymphocytic disorder, or a disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is a pulmonary disorder.In some embodiments, the pulmonary disorder is selected from the groupconsisting of asthma, airway hyperresponsiveness, and chronicobstructive pulmonary disease (COPD). In some embodiments, the pulmonarydisorder is asthma. In some embodiments, the asthma is Th2-high asthmaor Th2-low asthma. In some embodiments, the autoimmune disorder isselected from the group consisting of rheumatoid arthritis, psoriasis,eosinophilic esophagitis, inflammatory bowel disease (IBD), and Crohn'sdisease. In some embodiments, the inflammatory disorder is chronicidiopathic urticaria (CIU or CSU), anaphylaxis, anaphylactic shock,atopic dermatitis, or allergic rhinitis. In some embodiments, thefibrotic disorder is idiopathic pulmonary fibrosis (IPF). In someembodiments, the disorder is a disorder associated with increasednumbers or distribution of normal or aberrant tissue resident cells(such as mast cells, macrophages, or lymphocytes) or stromal cells (suchas fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is mastocytosis. In someembodiments, the method further comprises administering an additionaltherapeutic agent to the subject. In some embodiments, the additionaltherapeutic agent is an IL-13 axis binding antagonist, an IL-5 axisbinding antagonist, an IL-33 axis binding antagonist, an M1 primeantagonist, an IgE antagonist, a TRPA1 antagonist, a CRTH2 antagonist, abroncodilator or asthma symptom controller medication, animmunomodulator, a corticosteroid, a Th2 pathway inhibitor, a tyrosinekinase inhibitor, or a phosphodiesterase inhibitor. In some embodiments,the IL-13 axis binding antagonist is an anti-IL-13 antibody. In someembodiments, the anti-IL-13 antibody is lebrikizumab. In someembodiments, the IL-5 axis binding antagonist is an IL-5 bindingantagonist or an IL-5 receptor binding antagonist. In some embodiments,the IL-33 axis binding antagonist is an IL-33 binding antagonist or anST2 binding antagonist. In some embodiments, the IL-33 bindingantagonist is an anti-IL-33 antibody. In some embodiments, the M1 primeantagonist is quilizumab. In some embodiments, the IgE antagonist isomalizumab (Xolair®). In some embodiments, the antibody is administeredsubcutaneously, intravenously, intramuscularly, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. In someembodiments, the subject is a human.

In another aspect, the invention features a method of treating adisorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyone of the preceding compositions (e.g., pharmaceutical compositions).In some embodiments, the disorder is selected from the group consistingof a pulmonary disorder, an autoimmune disorder, an inflammatorydisorder, a fibrotic disorder, a granulocytic (neutrophilic oreosinophilic) disorder, a monocytic disorder, a lymphocytic disorder, ora disorder associated with increased numbers or distribution of normalor aberrant tissue resident cells (such as mast cells, macrophages, orlymphocytes) or stromal cells (such as fibroblasts, myofibroblasts,smooth muscle cells, epithelia, or endothelia). In some embodiments, thedisorder is a pulmonary disorder. In some embodiments, the pulmonarydisorder is selected from the group consisting of asthma, airwayhyperresponsiveness, and chronic obstructive pulmonary disease (COPD).In some embodiments, the pulmonary disorder is asthma. In someembodiments, the asthma is Th2-high asthma or Th2-low asthma. In someembodiments, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, psoriasis, eosinophilic esophagitis,inflammatory bowel disease (IBD), and Crohn's disease. In someembodiments, the inflammatory disorder is chronic idiopathic urticaria(CIU or CSU), anaphylaxis, anaphylactic shock, atopic dermatitis, orallergic rhinitis. In some embodiments, the fibrotic disorder isidiopathic pulmonary fibrosis (IPF). In some embodiments the disorder isa disorder associated with increased numbers or distribution of normalor aberrant tissue resident cells (such as mast cells, macrophages, orlymphocytes) or stromal cells (such as fibroblasts, myofibroblasts,smooth muscle cells, epithelia, or endothelia). In some embodiments, thedisorder is mastocytosis. In some embodiments, the method furthercomprises administering an additional therapeutic agent to the subject.In some embodiments, the additional therapeutic agent is an IL-13 axisbinding antagonist, an IL-5 axis binding antagonist, an IL-33 axisbinding antagonist, an M1 prime antagonist, an IgE antagonist, a TRPA1antagonist, a CRTH2 antagonist, a broncodilator or asthma symptomcontroller medication, an immunomodulator, a corticosteroid, a Th2pathway inhibitor, a tyrosine kinase inhibitor, or a phosphodiesteraseinhibitor. In some embodiments, the IL-13 axis binding antagonist is ananti-IL-13 antibody. In some embodiments, the anti-IL-13 antibody islebrikizumab. In some embodiments, the IL-5 axis binding antagonist isan IL-5 binding antagonist or an IL-5 receptor binding antagonist. Insome embodiments, the IL-33 axis binding antagonist is an IL-33 bindingantagonist or an ST2 binding antagonist. In some embodiments, the IL-33binding antagonist is an anti-IL-33 antibody. In some embodiments, theM1 prime antagonist is omalizumab (Xolair). In some embodiments, thecomposition is administered subcutaneously, intravenously,intramuscularly, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. In some embodiments, the subject isa human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment of the VH and VL domains of hu31A.v11 andhuE104.v2 showing the complementarity determining regions (CDRs)according to the Kabat, Chothia and Contact designations. Thehypervariable regions (HVRs) are underlined.

FIG. 2A is a series of graphs showing the results of an inhibitionanalysis of hu31A.v11 and huE104.v2 IgG as determined by a humantryptase enzymatic assay. Both antibodies completely inhibited tryptaseenzymatic activity.

FIGS. 2B and 2C are graphs showing the results of human primary airwaysmooth muscle cell (SMC) proliferation (FIG. 2B) and contraction (FIG.2C) assays. Addition of tryptase beta stimulated human primary airwaySMC proliferation, which was inhibited in a dose-dependent manner byaddition of the anti-tryptase antibody hu31A.v1 IgG4 or huE104.v2 IgG4(FIG. 2B). Addition of tryptase also stimulated human primary airway SMCcontraction, which was also inhibited by addition of hu31A.v11 IgG4 andhuE104.v2 IgG4 (FIG. 2C).

FIGS. 2D and 2E are graphs showing the results of mast celldegranulation assays in which mast cells were stimulated in vitro byaddition of tryptase beta or anti-4-hydroxy-3-nitrophenylacetyl (NP) IgEand NP. Addition of tryptase resulted in histamine release, which wasblocked by addition of hu31A.v11 (FIG. 2D). A catalytically-inactivemutant tryptase (S195A) served as a control. Addition of IgE and NP alsoresulted in histamine release, which was inhibited (30-50%) by additionof a tryptase small molecule inhibitor (SMI) or hu31A.v1 (FIG. 2E).

FIG. 3A is a graph showing the results of dissociation of human tryptasebeta 1 tetramer by hu31A.v1 Fab analyzed by size exclusionchromatography (SEC). Three runs were analyzed by SEC: run 1 containedWT tetrameric tryptase alone, which resulted in peak 1 with a retentiontime Tr=26 min, a retention volume Vr=13 ml; run 2 contained WTtetrameric tryptase+Fab hu31A.v11+heparin, which resulted in peak 2(Tr=27.6 min, Vr=13.8 ml) and peak 4 (Tr=31 min, Vr=15.5 ml); run 3contained WT tetrameric tryptase+Fab hu31A.v1 without heparin, whichresulted in peak 3 (Tr=28.1 min, Vr=14 ml) and peak 4 (Tr=31 min,Vr=15.5 ml).

FIG. 3B is a graph showing the results of dissociation of human tryptasebeta 1 tetramer by huE104.v2 Fab. Three runs were analyzed by SEC: run 1contained His-tagged monomeric tryptase+Fab huE104.v2, which resulted inpeak 2 (Tr=25.8 min) and peak 6 (31.6 min); run 2 contained WTtetrameric tryptase+Fab huE104.v2, which resulted in peak 3 (Tr=26 min)and peak 7 (Tr=31.8 min); and run 3 contained WT tetrameric tryptase+FabhuE104.v2+heparin, which resulted in peak 1 (Tr=21 min), peak 4 (Tr=27.2min) and peak 5 (Tr=31.2 min).

FIGS. 4A and 4B are series of graphs showing the results of apharmacokinetic (PK) simulation that compares the PK and neutralizingactivity of a dissociating anti-tryptase antibody with a tryptasetetramer-specific stabilizing antibody at a baseline tryptase level (4ng/ml serum, 10 ng/ml lung tissue, FIG. 4A) or a high tryptase level (10ng/ml serum, 40 ng/ml lung tissue, FIG. 4B).

FIG. 4C is a series of graphs showing the results of neutralizingactivity simulation comparing a dissociating anti-tryptase antibody anda tryptase tetramer-specific stabilizing antibody at the baselinetryptase level or a high tryptase level.

FIGS. 5A and 5B show images of Coomassie blue-stained sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (top panels),showing that human tryptase beta 1 cleaved fibrinogen into peptidefragments at both pH 6 (FIG. 5A) and 7.5 (FIG. 58). The anti-tryptaseantibody hu31A.v11 Fab blocked fibrinogen cleavage at both pH 6 and pH7.5, while huE102.v2 Fab did not, under the experimental conditions ofhigh concentration of heparin. Lane 1, fibrinogen only, showing thealpha, beta, and gamma chains of uncleaved fibrinogen; lane 2,fibrinogen and tryptase beta; lane 3, fibrinogen, tryptase beta, andhu31A.v1 Fab; lane 4, fibrinogen, tryptase beta, and B12 IgG; lane 5,fibrinogen, tryptase beta 1, and huE104.v2 Fab; and lane 6 shows B12mIgG1 alone. The decrease in intensity of the alpha chain indicatestryptase proteolytic activity, which was analyzed and quantified in thebottom panels.

FIG. 6A is a graph showing the results of dissociation of WT or mutanttetramer by hu31A.v11 Fab. Four runs were analyzed by SEC: run 1contained WT tetramer+huE104.v1 Fab, which resulted in the referencepeak (Tr=21.6 min); run 2 contained tetramer Y75C variant+hu31A.v11 Fab,which resulted in peak 1 (Tr=25.6 min) and peak 4 (Tr=31.6 min); run 3contained tetramer I99C variant+hu31A.v11 Fab, which resulted in peak 2(Tr=23.9 min) and peak 4 (Tr=31.6 min); and rune 4 contained WTtetramer+hu31A.v1 Fab, which resulted in peak 3 (Tr=28.1 min) and peak 4(Tr=31.6 min).

FIG. 6B shows the results of a Coomassie-blue stained SDS-PAGE gelanalysis of size exclusion chromatography peaks a FIG. 6A. hu31A.v11 Fabformed complexes with tryptase mutants Y75C and I99C and dissociated thetetramer into covalently linked dimers.

FIG. 6C is a graph showing the results of dissociation of WT or mutanttetramer by huE104.v2 Fab. Three runs were analyzed by SEC: run 1contained tetramer Y75C variant+huE104.v2 Fab, which resulted in peak 1(Tr=21.6 min) and peak 4 (Tr=31 min); run 2 contained tetramer I99Cvariant+huE104.v2 Fab, which resulted in peak 2 and peak 5 (Tr=31 min);and run 3 contained WT tetramer+huE104.v2 Fab, which resulted in peak 3(Tr=26 min) and peak 6 (Tr=31.8 min). The results show that huE104.v2Fab formed complexes with tryptase mutants Y75C and I99C and onlydissociated the I99C large interface-locked tetramer into covalentlylinked dimers. Peaks 4, 5 and 6 all contain excess Fab as determined bySDS-PAGD (data not shown).

FIG. 7 shows the amino acid sequence of mature human tryptase beta 1along with the gene sequential numbering and the chymotrypsinogennumbering (“chymo-numb”) system typically used for mammalian serinetrypsin.

FIG. 8 shows the binding epitope of Fab hu31A.v11 on human tryptase beta1 (tryptase residues all chymotrypsinogen numbering).

FIG. 9 is a rendering showing modeling of Fab hu31A.v11 onto thetryptase tetramer. The tryptase monomers in complex with Fab hu31A.v11were aligned to protomers A and C in the tryptase tetramer. Heavy chainsand light chains are indicated. Clashes between the light chains of Fabhu31A.v11 on adjacent tryptase protomers in this model are highlightedby a dashed oval.

FIG. 10 shows conformational changes in the 60s loop of tryptasedetected in the complex structure. Val60c and Val90 (shown in sticks)create a hydrophobic pocket for binding of Tyr173d from the neighboringprotomer as part of the protein-protein interaction in the largeinterface of tetrameric tryptase. Tryptase protomers in the tetramerconformation are indicated. Tryptase bound to Fab hu31A.v11 issuperposed to one protomer of the tetramer complex. The conformation ofVal60c changes when Fab hu31A.v11 is bound, which creates sterichindrance that is expected to prevent Tyr173d from binding to thatpocket (tryptase residues all chymotrypsinogen numbering).

FIG. 11 shows conformational changes in the 30s loop of tryptase locatedin the small interface detected in the complex structure after bindingto hu31A.v11.

FIG. 12A is a rendering of the crystal structure of WT tryptase tetramerin complex with four huE104.v1 Fabs. Tryptase protomers are indicatedaccording to the letter labeling or the protomers (see Pereira et al.Nature 392:306-311, 1998).

FIG. 12B is a rendering of the effect of huE104.v1 binding on the smallinterface of the tryptase tetramer as assessed by hydrogen-deuteriumexchange (HDX).

FIG. 13 is a graph showing the results of a pharmacokinetic (PK)analysis of humanized anti-tryptase antibodies huE104.v2 and hu31A.v1,each administered by intravenous (IV) injection at 1 or 10 mg/kg toC57BL/6 mice. The graph shows anti-tryptase antibody concentration(pg/mL) as a function of time (days). The results are from threeanimals.

FIG. 14 is a graph showing the results of a PK analysis of humanizedanti-tryptase antibodies huE104.v2 and hu31A.v1 compared to a controlanti-gD IgG4 antibody. Each antibody was administered by IV injection at30 mg/kg to cynomolgus (cyno) monkeys. The graph shows anti-tryptaseantibody concentration (pg/mL) as a function of time (days).

FIG. 15 is a schematic diagram of assays for measuring the amount ofactive tryptase (left panel) and total tryptase (right panel) in asample. Tryptase monomers and tetramers are indicated. In the leftpanel, soy bean trypsin inhibitor (SBTI) is added. Next, an exemplarybiotinylated activity-based probe (ABP) was added to atryptase-containing sample (e.g., broncoalveolar lavage fluid (BAL)) tolabel active tryptase. Labeling was stopped by addition of an exemplarysmall molecule inhibitor G02849855 (e.g., BMS-262084, Sutton et al.Bioorg. Med. Chem. Lett. 12:3229-33, 2002, Qian et al., J. Org. Chem.2002, 67:3595-3600). The hu31A.v1 antibody was added to dissociate thetryptase tetramers. The labeled tryptase was then detected in anenzyme-linked immunosorbent assay (ELISA) using horseradish peroxidaseconjugated to streptavidin. In the total tryptase assay, hu31A.v11 wasadded to a sample to dissociate tryptase tetramers in the sample. Theamount of tryptase was then determined using ELISA.

FIG. 16 shows the results of an active tryptase assay performed on BALsamples obtained from cyno monkeys that were administered theanti-tryptase antibody hu31A.v1 by intravenous (IV) administration at 30mg/kg. The top panel shows a schematic diagram of the experimentalprotocol.

FIG. 17 is a schematic diagram showing the experimental protocol of thecyno Ascaris challenge model described in Example 6.

FIGS. 18A and 18B are graphs showing the results of an active tryptaseassay (FIG. 18A) and a total tryptase assay (FIG. 18B) performed on BALobtained from individual animals in the cyno Ascaris challengeexperiment described in Example 6.

FIG. 18C is a graph showing the results of a total tryptase assay todetermine the amount of total tryptase in nasal mucosal lining fluid(MLF) obtained by nasosorption using a synthetic absorptive matrix (SAM)from individual animals in the cyno Ascaris challenge experimentdescribed in Example 6.

FIG. 19 is a graph showing that administration of the anti-tryptaseantibody hu31A.v11 inhibited IgE-mediated passive systemic anaphylaxisin human engrafted mice. Upon IgE challenge, mice treated with theanti-tryptase antibody hu31A.v11 showed an improved body temperaturemaintenance as compared to mice treated with a control anti-gD antibody.*** P<0.0001 (Paired T test).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

The term “K_(D) is measured by a surface plasmon resonance assay,” whenused in the context of the claims, means that the K_(D) is measuredaccording the method described in Example 1(A)(vii), which measureskinetic parameters for binding of anti-tryptase antibodies to humantryptase beta 1 monomer, e.g., His6-tagged tryptase monomer as shown inSEQ ID NO: 128, which does not spontaneously form a tryptase tetramer.The assay can use a BIAcore® T200 or an equivalent device. Briefly,BIAcore® Series S CM5 sensor chips (or equivalent sensor chips) areimmobilized with monoclonal mouse anti-human IgG (Fc) antibody andanti-tryptase antibodies are subsequently captured on the flow cell.Serial 3-fold dilutions of the human tryptase beta 1 monomer areinjected at a flow rate of 30 μl/min. Each sample is analyzed with 3 minassociation and 10 min dissociation. The assay is performed at 25° C.After each injection, the chip is regenerated using 3 M MgCl₂. Bindingresponse is corrected by subtracting the response units (RU) from a flowcell capturing an irrelevant IgG at similar density. A 1:1 Languir modelof simultaneous fitting of k_(on) and k_(off) is used for kineticsanalysis.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs and/or framework regions which result in an improvementin the affinity of the antibody for antigen, compared to a parentantibody which does not possess those alteration(s). Preferredaffinity-matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity-matured antibodies areproduced by procedures known in the art. For example, Marks et al.Bio/Technology 10:779-783, 1992 describes affinity maturation by VH andVL domain shuffling. Random mutagenesis of HVR and/or framework residuesis described by: Barbas et al. Proc. Nat. Acad. Sci. USA 91:3809-3813,1994; Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol.155:1994-2004, 1995; Jackson et al. J. Immunol. 154(7):3310-3319, 1995;and Hawkins et al. J. Mol. Biol. 226:889-896, 1992.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

As used herein, “tryptase” refers to any native tryptase from anyvertebrate source, including mammals such as primates (e.g., humans) androdents (e.g., mice and rats), unless otherwise indicated. Tryptase isalso known in the art as mast cell tryptase, mast cell protease II, skintryptase, lung tryptase, pituitary tryptase, mast cell neutralproteinase, and mast cell serine proteinase II. The term “tryptase”encompasses tryptase alpha (encoded in humans by TPSAB1), tryptase beta(encoded in humans by TPSAB1 and TPSB2; see below), tryptase delta(encoded in humans by TPSD1), tryptase gamma (encoded in humans byTPSG1), and tryptase epsilon (encoded in humans by PRSS22). Tryptasealpha, beta, and gamma proteins are soluble, whereas tryptase epsilonproteins are membrane anchored. Tryptase beta and gamma are activeserine proteases, although they have different specificities. Tryptasealpha and delta proteins are largely inactive proteases as they haveresidues in critical position that differ from typical active serineproteases. An exemplary tryptase alpha full length protein sequence canbe found under NCBI GenBank Accession No. ACZ98910.1 (SEQ ID NO: 118).Exemplary tryptase gamma full length protein sequences can be foundunder Uniprot Accession No. Q9NRR2 or GenBank Accession Nos. Q9NRR2.3,AAF03695.1, NP_036599.3 or AAF76457.1. Exemplary tryptase delta fulllength protein sequences can be found under Uniprot Accession No. Q9BZJ3or GenBank Accession No. NP_036349.1. Several tryptase genes areclustered on human chromosome 16p13.3. The term encompasses“full-length,” unprocessed tryptase as well as any form of tryptase thatresults from processing in the cell. Tryptase beta is the main tryptaseexpressed in mast cells, while tryptase alpha is the main tryptaseexpressed in basophils. Tryptase alpha and tryptase beta typicallyinclude a leader sequence of approximately 30 amino acids and acatalytic sequence of approximately 245 amino acids (see, e.g.,Schwartz, Immunol. Allergy Clin. N. Am. 26:451-463, 2006).

As used herein, “tryptase beta” refers to any native tryptase beta fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats), unless otherwise indicated. Tryptasebeta is a serine protease that is a major constituent of mast cellsecretory granules. As used herein, the term encompasses tryptase beta 1(encoded by the TPSAB1 gene, which also encodes tryptase alpha 1),tryptase beta 2 (encoded by the TPSB2 gene), and tryptase beta 3 (alsoencoded by the TPSB2 gene). An exemplary human tryptase beta 1 sequenceis shown in SEQ ID NO: 71 (see also GenBank Accession No. NP_003285.2).An exemplary human tryptase beta 2 sequence is shown in SEQ ID NO: 72(see also GenBank Accession No. AAD13876.1). An exemplary human tryptasebeta 3 sequence is shown in SEQ ID NO: 73 (see also GenBank AccessionNo. NP_077078.5). The term tryptase beta encompasses “full-length,”unprocessed tryptase beta as well as tryptase beta that results frompost-translational modifications, including proteolytic processing.Full-length, pro-tryptase beta is thought to be processed in twoproteolytic steps. First, autocatalytic intermolecular cleavage atR-occurs, particularly at acidic pH and in the presence of a polyanion(e.g., heparin or dextran sulfate). Next, the remaining pro′ dipeptideis removed (likely by dipeptidyl peptidase I). For full-length humantryptase beta 1, with reference to SEQ ID NO: 71 below, the underlinedamino acid residues correspond to the native leader sequence, and thebolded and gray-shaded amino acid residues correspond to the pro-domain,which are cleaved to form the mature protein (see, e.g., Sakai et al. J.Clin. Invest. 97:988-995, 1996)

(SEQ ID NO: 71)

GSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKP.Mature, enzymatically active tryptase beta is typically a homotetrameror heterotetramer, although active monomer has been reported (see, e.g.,Fukuoka et al. J. Immunol. 176:3165, 2006). The subunits of the tryptasebeta tetramer are held together by hydrophobic and polar interactionsbetween subunits and stabilized by polyanions (particularly heparin anddextran sulfate). The term tryptase can refer to tryptase tetramer ortryptase monomer. Exemplary sequences for mature human tryptase beta 1,beta 2, and beta 3 are shown in SEQ ID NO: 97, SEQ ID NO: 116, and SEQID NO: 117, respectively. The active site of each subunit faces into acentral pore of the tetramer, which measures approximately 50×30angstroms (see, e.g., Pereira et al. Nature 392:306-311, 1998). The sizeof the central pore typically restricts access of the active sites byinhibitors. Exemplary substrates of tryptase beta include, but are notlimited to, PAR2, C3, fibrinogen, fibronectin, and kininogen.

The terms “anti-tryptase antibody,” an “antibody that binds totryptase,” and “antibody that specifically binds tryptase” refer to anantibody that is capable of binding tryptase with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting tryptase. In one embodiment, the extent of binding ofan anti-tryptase antibody to an unrelated, non-tryptase protein is lessthan about 10% of the binding of the antibody to tryptase as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to tryptase has a dissociation constant (K_(D)) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M orless, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-tryptase antibody binds to an epitope oftryptase that is conserved among tryptase from different species.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that contacts an overlapping set of amino acidresidues of the antigen as compared to the reference antibody or blocksbinding of the reference antibody to its antigen in a competition assayby 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.In some embodiments, the set of amino acid residues contacted by theantibody may be completely overlapping or partially overlapping with theset of amino acid residues contacted by the reference antibody. In someembodiments, an antibody that binds to the same epitope as a referenceantibody blocks binding of the reference antibody to its antigen in acompetition assay by 50% or more, 60% or more, 70% or more, 80% or more,or 90% or more, and conversely, the reference antibody blocks binding ofthe antibody to its antigen in a competition assay by 50% or more, 60%or more, 70% or more, 80% or more, or 90% or more. An exemplarycompetition assay is provided herein.

The term “is determined by an epitope binning assay,” in the context ofthe claims, means that an antibody is determined to bind to the sameepitope and/or compete for binding with a reference anti-tryptaseantibody (e.g., hu31A.v11 or huE104.v2) using the OCTET® epitope binningassay such as described in Example 3, Section C. Briefly, human tryptasebeta 1 monomer protein is biotinylated at Lys residue by reacting withNHS-PEG4-biotin. Biotinylated monomer is diluted to 5 μg/ml in kineticsbuffer (ForteBio, Inc.) and immobilized onto streptavidin sensor tips(ForteBio, Inc.). After the immobilization step, human tryptase beta1-immobilized sensors are saturated with the first antibody, diluted at10-20 μg/ml, followed by binding with second antibody diluted at 2.5μg/ml. The run temperature for such epitope binding assays is 30° C. Abinding signal by second antibody implies that the two antibodies canbind antigen simultaneously at distinct, non-overlapping epitopes,whereas no binding signal implies that they share a common epitope. Insome instances, a partial signal by the second antibody is observed(i.e., the signal is less than the signal observed if the first antibodywas not added, but greater than background), which implies the epitopesare partially overlapping.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al. Protein Eng. 8(10):1057-1062, 1995);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (VH), and the first constant domain of one heavychain (C_(H)1). Pepsin treatment of an antibody yields a single largeF(ab′)2 fragment which roughly corresponds to two disulfide linked Fabfragments having divalent antigen-binding activity and is still capableof cross-linking antigen. Fab′ fragments differ from Fab fragments byhaving an additional few residues at the carboxy terminus of the C_(H)1domain including one or more cysteines from the antibody hinge region.Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only three Hsspecific for an antigen) has the ability to recognize and bind antigen,although often at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315, 1994.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.

By “binding domain” is meant a part of a compound or a molecule thatspecifically binds to a target epitope, antigen, ligand, or receptor.Binding domains include but are not limited to antibodies (e.g.,monoclonal, polyclonal, recombinant, humanized, and chimericantibodies), antibody fragments or portions thereof (e.g., Fabfragments, Fab′2, scFv antibodies, SMIP, domain antibodies, diabodies,minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains ofantibodies), receptors, ligands, aptamers, and other molecules having anidentified binding partner.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen. In some embodiments, theactivity may be a tryptase enzymatic activity, e.g., protease activity.In other instances, the activity may be tryptase-mediated stimulation ofbronchial smooth muscle cell proliferation and/or collagen-basedcontraction. In other instances, the activity may be mast cell histaminerelease (e.g., IgE-triggered histamine release and/or tryptase-triggeredhistamine release). An antibody of the invention can inhibit abiological activity of tryptase at least about 1%, about 5%, about 10%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100%.

The term “as determined by a human tryptase beta enzymatic assay using asynthetic peptide S-2288 as a substrate,” in the context of the claims,means that inhibitory activity is measured according to the assaydescribed in Example 1(A)(viii)(a). Briefly, recombinant human tryptasebeta 1 tetramer active enzyme is diluted to 0.75 nM in TNH Buffer (200mM Tris, 150 mM NaCl, 0.1 mg/mL heparin, 0.01% TRITON™ X-100, pH 8.0),and combined 1:1 with anti-tryptase antibodies (diluted in PBS) in384-well plates. Plates are incubated for 1 h at ambient temperaturewith gentle agitation. Colorimetric substrate S-2288 (Chromogenix, PartNo. 82-0852-39), or an equivalent substrate, is diluted to 1200 μM inTNH Buffer and added to the plate. Final in-well concentrations are 400μM-2288, 0.25 nM recombinant human tryptase beta 1 tetramer, 66 μg/mLheparin, and from 0.10 to 222 nM anti-tryptase antibody. Plates areincubated for 40 min at ambient temperature with gentle agitation andthen read at A₄₀₅. The IC50 of the anti-tryptase antibodies isdetermined from a four-parameter fit of their respective curves.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoieticcells is summarized in Table 3 on page 464 of Ravetch et al. Annu. Rev.Immunol. 9:457-492, 1991. To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 can be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest can be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA95:652-656,1998.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRI receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234,1997). FcRs are reviewed, forexample, in Ravetch et al. Annu. Rev. Immunol. 9:457-492,1991; Capel etal. Immunomethods 4:25-34,1994; and de Haas et al. J. Lab. Clin. Med.126:330-41, 1995. Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (see, e.g., Guyer et al. J. Immunol. 117:587,1976; and Kim et al. J. Immunol. 24:249, 1994).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils; with PBMCs and NK cells being preferred. The effector cellscan be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al. J.Immuno. Methods 202:163, 1996, can be performed.

An “epitope” is the portion of the antigen to which the antibodyselectively binds. For a polypeptide antigen, a linear epitope can be apeptide portion of about 4-15 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, aminoacid residues. A non-linear, conformational epitope may compriseresidues of a polypeptide sequence brought to close vicinity in thethree-dimensional (3D) structure of the protein. In some embodiments,the epitope comprises amino acids that are within 4 angstroms (Å) of anyatom of an antibody. In some embodiments, the epitope comprises aminoacids of a tryptase protomer that are within 4 Å of any atom of apartner Fab. In certain embodiments, the epitope comprises amino acidsthat are within 3.5 Å, 3 Å, 2.5 Å, or 2 Å of any atom of an antibody.The amino acid residues of an antibody that contact an antigen (i.e.,paratope) can be determined, for example, by determining the crystalstructure of the antibody in complex with the antigen or by performinghydrogen/deuterium exchange.

The term “the epitope is determined by an X-ray crystallography model,”when used in the context of the claims, means that an atom of a tryptaseamino acid residue (e.g., human tryptase beta 1 residue) is determinedto be within 4 Å of any atom of an anti-tryptase antibody (e.g., anyanti-tryptase antibody described herein, e.g., hu31A.v11 or huE104.v2)in an X-ray crystallography model, for example, as described in Example3. In some embodiments, the X-ray crystallography model has a resolutionof about 3.5 Å or less, about 3 Å or less, about 2.5 Å or less, about2.15 Å or less, or about 2 Å or less.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies. Thisdefinition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md., vols. 1-3, 1991. In oneembodiment, for the VL, the subgroup is subgroup kappa III or kappa IVas in Kabat et al. supra. In one embodiment, for the VH, the subgroup issubgroup III as in Kabat et al. supra.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al. Nature321:522-525, 1986; Riechmann et al. Nature 332:323-329, 1988; andPresta, Curr. Op. Struct. Biol. 2:593-596, 1992.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “isolated” when used to describe the various antibodiesdisclosed herein, means an antibody that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC) methods. For a review of methods for assessmentof antibody purity, see, for example, Flatman et al. J. Chromatogr. B848:79-87, 2007. In preferred embodiments, the antibody will be purified(1) to a degree sufficient to obtain at least 15 residues of N-terminalor internal amino acid sequence by use of a spinning cup sequenator, or(2) to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes antibodies in situ within recombinant cells, because at leastone component of the polypeptide natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope on an antigen, except for possible variantantibodies, e.g., containing naturally occurring mutations or arisingduring production of a monoclonal antibody preparation, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. Thus, the modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein. In certain embodiments, the term “monoclonal antibody”encompasses bispecific antibodies.

The term “bivalent antibody” refers to an antibody that has two bindingsites for the antigen. A bivalent antibody can be, without limitation,in the IgG format or in the F(ab′)₂ format.

The term “multispecific antibody” is used in the broadest sense andcovers an antibody that binds to two or more determinants or epitopes onone antigen or two or more determinants or epitopes on more than oneantigen. Such multispecific antibodies include, but are not limited to,full-length antibodies, antibodies having two or more VL and VH domains,antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecificdiabodies and triabodies, antibody fragments that have been linkedcovalently or non-covalently. “Polyepitopic specificity” refers to theability to specifically bind to two or more different epitopes on thesame or different target(s). In certain embodiments, the multispecificantibody is a bispecific antibody. “Dual specificity” or “bispecificity”refers to the ability to specifically bind to two different epitopes onthe same or different target(s). However, in contrast to bispecificantibodies, dual-specific antibodies have two antigen-binding arms thatare identical in amino acid sequence and each Fab arm is capable ofrecognizing two antigens. Dual-specificity allows the antibodies tointeract with high affinity with two different antigens as a single Fabor IgG molecule. According to one embodiment, the multispecific antibodybinds to each epitope with an affinity of 5 pM to 0.001 pM, 3 pM to0.001 pM, 1 pM to 0.001 pM, 0.5 pM to 0.001 pM or 0.1 pM to 0.001 pM.“Monospecific” refers to the ability to bind only one epitope.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical composition.

With regard to the binding of a antibody to a target molecule, the term“binds” or “binding” or “specific binding” or “specifically binds” or is“specific for” a particular polypeptide or an epitope on a particularpolypeptide target means binding that is measurably different from anon-specific interaction. Specific binding can be measured, for example,by determining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a K_(D) forthe target of 10⁻⁴ M or lower, alternatively 10⁻⁵ M or lower,alternatively 10⁻⁶ M or lower, alternatively 10⁻⁷ M or lower,alternatively 10⁻⁸ M or lower, alternatively 10⁻⁹ M or lower,alternatively 10⁻¹⁰ M or lower, alternatively 10⁻¹¹ M or lower,alternatively 10⁻¹² M or lower or a K_(D) in the range of 10⁻⁴ M to 10⁻⁶M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹ M. As will be appreciated bythe skilled artisan, affinity and K_(D) values are inversely related. Ahigh affinity for an antigen is measured by a low K_(D) value. In oneembodiment, the term “specific binding” refers to binding where amolecule binds to a particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

By “paratope” is meant the part of an antibody which binds the epitopeof an antigen. The paratope is typically a region of about 15-22 aminoacid residues of the antibody's Fv region and may contain amino acidsfrom the antibody's V_(H) and V_(L) chains.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. Thevariable or “V” domain mediates antigen binding and defines specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the 110-amino acid span ofthe variable domains. Instead, the V regions consist of relativelyinvariant stretches called framework regions (FRs) of 15-30 amino acidsseparated by shorter regions of extreme variability called“hypervariable regions” that are each 9-12 amino acids long. The term“hypervariable region” or “HVR” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues frome.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in theVL, and around about residues 26-35 (H1), 49-65 (H2) and 95-102 (H3) inthe VH (in one embodiment, H1 is around about residues 31-35); Kabat etal. supra) and/or those residues from a “hypervariable loop” (e.g.,residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the VL, and 26-32(H1), 53-55 (H2), and 96-101 (H3) in the VH; Chothia et al. J. Mol.Biol. 196:901-917, 1987. The variable domains of native heavy and lightchains each comprise four FRs, largely adopting a beta-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the beta-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al. supra). Accordingly, the HVR and FRsequences generally appear in the following sequence in VH (or VL):FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al. supra.Using this numbering system, the actual linear amino acid sequence maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or HVR of the variable domain. For example,a heavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al. supra). The“EU numbering system” or “EU index” is generally used when referring toa residue in an immunoglobulin heavy chain constant region (e.g., the EUindex reported in Kabat et al. supra). The “EU index as in Kabat” refersto the residue numbering of the human IgG1 EU antibody. Unless statedotherwise herein, references to residue numbers in the variable domainof antibodies means residue numbering by the Kabat numbering system.Unless stated otherwise herein, references to residue numbers in theconstant domain of antibodies means residue numbering by the EUnumbering system (e.g., see U.S. Provisional Application No. 60/640,323,Figures for EU numbering).

A “disorder” or “disease” is any condition that would benefit fromtreatment with the antibody (e.g., any anti-tryptase antibody describedherein). This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. In some embodiments, the disorder is a pulmonarydisorder, an autoimmune disorder, an inflammatory disorder, a fibroticdisorder, a granulocytic (neutrophilic or eosinophilic) disorder, amonocytic disorder, a lymphocytic disorder, or a disorder associatedwith increased numbers or distribution of normal or aberrant tissueresident cells (such as mast cells, macrophages, or lymphocytes) orstromal cells (such as fibroblasts, myofibroblasts, smooth muscle cells,epithelia, or endothelia). In some embodiments, the disorder is apulmonary disorder. In some examples, a disorder may be atryptase-associated disorder or a tryptase-mediated disorder.

The terms “tryptase-associated disorder” and “tryptase-mediateddisorder,” as used herein, refers to any disorder or condition mediatedby, or associated with, tryptase. In some embodiments,tryptase-associated disorders are associated with excess tryptase levelsor activity in which atypical symptoms may manifest due to the levels oractivity of tryptase locally and/or systemically in the body.

In some embodiments, the pulmonary disorder is asthma. In someembodiments, the asthma is persistent chronic severe asthma with acuteevents of worsening symptoms (exacerbations or flares) that can be lifethreatening. In some embodiments, the asthma is atopic (also known asallergic) asthma, non-allergic asthma (e.g., often triggered byinfection with a respiratory virus (e.g., Influenza, parainfluenza,rhinovirus, human metapneumovirus, and respiratory syncytial virus) orinhaled irritant (air pollutants, smog, diesel particles, volatilechemicals and gases indoors or outdoors, or even by cold dry air).

In some embodiments, the asthma is intermittent or exercise-induced,asthma due to acute or chronic primary or second-hand exposure to“smoke” (typically cigarettes, cigars, pipes), inhaling or “vaping”(tobacco, marijuana or other such substances), or asthma triggered byrecent ingestion of aspirin or related NSAIDS. In some embodiments, theasthma is mild, or corticosteroid naïve asthma, newly diagnosed anduntreated asthma, or not previously requiring chronic use of inhaledtopical or systemic steroids to control the symptoms (cough, wheeze,shortness of breath/breathlessness, or chest pain). In some embodiments,the asthma is chronic, corticosteroid resistant asthma, corticosteroidrefractory asthma, asthma uncontrolled on corticosteroids or otherchronic asthma controller medications.

In some embodiments, the asthma is moderate to severe asthma. In certainembodiments, the asthma is Th2-high asthma. In some embodiments, theasthma is severe asthma. In some embodiments, the asthma is atopicasthma, allergic asthma, non-allergic asthma (e.g., due to infectionand/or respiratory syncytial virus (RSV)), exercise-induced asthma,aspirin sensitive/exacerbated asthma, mild asthma, moderate to severeasthma, corticosteroid naïve asthma, chronic asthma, corticosteroidresistant asthma, corticosteroid refractory asthma, newly diagnosed anduntreated asthma, asthma due to smoking, asthma uncontrolled oncorticosteroids. In some embodiments, the asthma is T helper lymphocytetype 2 (Th2) or type 2 (Th2) high, or Type 2 (T2)-driven asthma. In someembodiments, the asthma is eosinophilic asthma. In some embodiments, theasthma is allergic asthma. In some embodiments, the individual has beendetermined to be Eosinophilic Inflammation Positive (EIP). See WO2015/061441. In some embodiments, the asthma is periostin-high asthma(e.g., having periostin level at least about any of 20 ng/mL, 25 ng/mL,or 50 ng/mL serum). In some embodiments, the asthma is eosinophil-highasthma (e.g., at least about any of 150, 200, 250, 300, 350, 400eosinophil counts/ml blood). In certain embodiments, the asthma isTh2-low asthma or nonTh2-driven asthma. In some embodiments, theindividual has been determined to be Eosinophilic Inflammation Negative(EIN). See WO 2015/061441. In some embodiments, the asthma isperiostin-low asthma (e.g., having periostin level less than about 20ng/mL serum). In some embodiments, the asthma is eosinophil-low asthma(e.g., less than about 150 eosinophil counts/μl blood or less than about100 eosinophil counts/μl blood).

The term “Th2-high asthma,” as used herein, refers to asthma thatexhibits high levels of one or more Th2 cell-related cytokines, forexample, IL13, IL4, IL9, IL5, or that exhibits Th2 cytokine-associatedinflammation. In certain embodiments, the term Th2-high asthma may beused interchangeably with eosinophil-high asthma. In certainembodiments, the Th2-high asthma is Th2 driven asthma. In someembodiments, the asthma patient has been determined to be EosinophilicInflammation Positive (EIP). See, e.g., International Patent ApplicationPublication No. WO 2015/061441, which is incorporated by referenceherein in its entirety. In certain embodiments, the individual has beendetermined to have elevated levels of at least one of the eosinophilicsignature genes as compared to a control or reference level. SeeWO2015/061441. In certain embodiments, the Th2-high asthma isperiostin-high asthma. In some embodiments, the individual has highserum periostin. In certain embodiments, the individual is eighteenyears or older. In certain embodiments, the individual has beendetermined to have an elevated level of serum periostin as compared to acontrol or reference level. In certain embodiments, the control orreference level is the median level of periostin in a population. Incertain embodiments, the individual has been determined to have 20 ng/mlor higher serum periostin. In certain embodiments, the individual hasbeen determined to have 25 ng/ml or higher serum periostin. In certainembodiments, the individual has been determined to have 50 ng/ml orhigher serum periostin. In certain embodiments, the control or referencelevel of serum periostin is 20 ng/ml, 25 ng/ml, or 50 ng/ml. In certainembodiments, the asthma is eosinophil-high asthma. In certainembodiments, the individual has been determined to have an elevatedeosinophil count as compared to a control or reference level. In certainembodiments, the control or reference level is the median level of apopulation. In certain embodiments, the individual has been determinedto have 150 or higher eosinophil count/μl blood. In certain embodiments,the individual has been determined to have 200 or higher eosinophilcount/μl blood. In certain embodiments, the individual has beendetermined to have 250 or higher eosinophil count/μl blood. In certainembodiments, the individual has been determined to have 300 or highereosinophil count/μl blood. In certain embodiments, the individual hasbeen determined to have 350 or higher eosinophil count/μl blood. Incertain embodiments, the individual has been determined to have 400 orhigher eosinophil count/μl blood. In certain embodiments, the individualhas been determined to have 450 or higher eosinophil count/μl blood. Incertain embodiments, the individual has been determined to have 500 orhigher eosinophil count/μl blood. In certain preferred embodiments, theindividual has been determined to have 300 or higher eosinophil count/μlblood. In certain embodiments, the eosinophils are peripheral bloodeosinophils. In certain embodiments, the eosinophils are sputumeosinophils. In certain embodiments, the individual exhibits elevatedlevel of FeNO (fractional exhaled nitric acid) and/or elevated level ofIgE. For example, in some instances, the individual exhibits a FeNOlevel above about 250 parts per billion (ppb), above about 275 ppb,above about 300 ppb, above about 325 ppb, above about 325 ppb, or aboveabout 350 ppb. In some instances, the individual has an IgE level thatis above 50 IU/ml.

The term “Th2-low asthma” or “non-Th2-high asthma” as used herein,refers to asthma that exhibits low levels of one or more Th2cell-related cytokines, for example, IL13, IL4, IL9, IL5, or exhibitsnon-Th2 cytokine-associated inflammation. In certain embodiments, theterm Th2-low asthma may be used interchangeably with eosinophil-lowasthma. In some embodiments, the asthma patient has been determined tobe Eosinophilic Inflammation Negative (EIN). See, e.g., WO 2015/061441.In certain embodiments, the Th2-low asthma is Th17-driven asthma. Incertain embodiments, the Th2-low asthma is periostin-low asthma. Incertain embodiments, the individual is eighteen years or older. Incertain embodiments, the individual has been determined to have areduced level of serum periostin as compared to a control or referencelevel. In certain embodiments, the control or reference level is themedian level of periostin in a population. In certain embodiments, theindividual has been determined to have less than 20 ng/ml serumperiostin. In certain embodiments, the asthma is eosinophil-low asthma.In certain embodiments, the individual has been determined to have areduced esosinophil count as compared to a control or reference level.In certain embodiments, the control or reference level is the mediumlevel of a population. In certain embodiments, the individual has beendetermined to have less than 150 eosinophil count/μl blood. In certainembodiments, the individual has been determined to have less than 100eosinophil count/μl blood. In certain preferred embodiments, theindividual has been determined to have less than 300 eosinophil count/μlblood.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder is esophagitis (e.g.,eosinophilic esophagitis), allergic rhinitis, non-allergic rhinitis,rhinosinusitis with polyps, nasal polyposis, bronchitis, chronicpneumonia, allergic bronchopulmonary aspergillosis, airway inflammation,allergic rhinitis, bronchiectasis, and/or chronic bronchitis.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder, is arthritis. In someembodiments, the arthritis is rheumatoid arthritis. In some embodiments,the arthritis is osteoarthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, early arthritis, polyarticularrheumatoid arthritis, systemic-onset rheumatoid arthritis, enteropathicarthritis, reactive arthritis, psoriatic arthritis, and/or arthritis asa result of injury.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder is a gastrointestinalinflammatory condition. In some embodiments, the gastrointestinalinflammatory condition is IBD (inflammatory bowel disease), ulcerativecolitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused byenvironmental insults (e.g., caused by or associated with a therapeuticregimen, such as chemotherapy, radiation therapy, etc.)), infectiouscolitis, ischemic colitis, collagenous or lymphocytic colitis,necrotizing enterocolitis, colitis in conditions such as chronicgranulomatous disease or celiac disease, food allergies, gastritis,gastroenteritis, infectious gastritis or enterocolitis (e.g.,Helicobacter pylori-infected chronic active gastritis), esophagitis, andother forms of gastrointestinal inflammation caused by an infectiousagent, or indeterminate colitis.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder is a gastrointestinalinflammatory condition. In some embodiments, the gastrointestinalinflammatory condition is IBD (inflammatory bowel disease). In someembodiments the inflammatory bowel disease is ulcerative colitis (UC) orCrohn's disease (CD). In some embodiments, the gastrointestinalinflammatory condition is colitis (e.g., colitis caused by environmentalinsults (e.g., caused by or associated with a therapeutic regimen, suchas chemotherapy, radiation therapy, etc.), infectious colitis, ischemiccolitis, collagenous or lymphocytic colitis, necrotizing enterocolitis,colitis in conditions such as chronic granulomatous disease or celiacdisease, food allergies, gastritis, gastroenteritis, infectiousgastritis or enterocolitis (e.g., Helicobacter pylori-infected chronicactive gastritis), and other forms of gastrointestinal inflammationcaused by an infectious agent, or indeterminate colitis. In someembodiments, the gastrointestinal inflammatory condition is ulcerativecolitis (UC) or Crohn's disease (CD). In some embodiments, thegastrointestinal inflammatory condition is ulcerative colitis (UC). Insome embodiments, the ulcerative colitis is mild to moderate distalcolitis. In some embodiments, the ulcerative colitis is mild to moderateextensive colitis. In some embodiments, the ulcerative colitis is severecolitis. In some embodiments, the gastrointestinal inflammatorycondition is Crohn's disease (CD). In some embodiments, the Crohn'sdisease is in acute disease stage. In some embodiments, the Crohn'sdisease is in induced clinical remission stage. In some embodiments, theCrohn's disease is in maintain response/remission stage. In someembodiments, the Crohn's disease is mild to moderate disease. In someembodiments, the Crohn's disease is moderate to severe disease. In someembodiments, the Crohn's disease is severe/fulminant disease. In someembodiments, the Crohn's disease is ileal, ileocolonic, or colonicdisease.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder, or disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia) is lupus or Systemic Lupus Erythematosus (SLE), or one ormore organ-specific manifestations of lupus (e.g., lupus nephritis (LN)affecting the kidney, or extra-renal lupus (ERL) affecting the bloodand/or lymphoid organs (lymph nodes, spleen, thymus, and associatedlymphatic vessels), and/or joints and/or other organs, but notnecessarily the kidney).

In some embodiments, the autoimmune disorder, inflammatory disorder, orfibrotic disorder is related to sepsis and/or trauma, HIV infection, oridiopathic (of unknown etiology) such as ANCA-associated vaculitides(AAV), granulomatosis with polyangiitis (formerly known as Wegener'sgranulomatosis), Behcet's disease, cardiovascular disease, eosinophilicbronchitis, Reiter's Syndrome, SEA Syndrome (Seronegativity,Enthesopathy, Arthropathy Syndrome), ankylosing spondylitis,dermatomyositis, scleroderma, e.g., systemic scleroderma also calledsystemic sclerosis, vasculitis (e.g., Giant Cell Arteritis (GCA), alsocalled temporal arteritis, cranial arteritis or Horton disease),myositis, polymyositis, dermatomyositis, polyarteritis nodosa,arteritis, polymyalgia rheumatica, sarcoidosis, primary biliarysclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaquepsoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis,erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus, e.g.,pemphigus vulgaris, atherosclerosis, lupus, Still's disease, myastheniagravis, celiac disease, multiple sclerosis (MS) of therelapsing-remitting (RRMS) or primary progressive (PPMS) or secondaryprogressive (SPMS) subtypes, Guillain-Barre disease, Type I diabetesmellitus (T1DM) or insulin-dependent (IDDM) or juvenile onset DM type,thyroiditis (e.g., Graves' disease), coeliac disease, Churg-Strausssyndrome, myalgia syndrome, hypereosinophilic syndrome, oedematousreactions including episodic angioedema, helminth infections,onchocercal dermatitis, eosinophilic esophagitis, eosinophilicenteritis, eosinophilic colitis, obstructive sleep apnea, endomyocardialfibrosis, Addison's disease, Raynaud's disease or phenomenon, autoimmunehepatitis, graft versus host disease (GVHD), or organ transplantrejection.

In some embodiments, the disorder is an inflammatory disorder of theskin. In some embodiments, the disorder is atopic dermatitis oronchocercal dermatitis. In some embodiments, the disorder is chronicidiopathic urticaria (CIU or CSU).

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, neutrophilic disorder, or eosinophilic disorder is afibrotic disorder. In some embodiments, the fibrotic disorders includelung fibrosis, liver fibrosis (e.g., fibrosis associated with cirrhosis(e.g., alcohol-induced cirrhosis, viral-induced cirrhosis,post-hepatitis C cirrhosis, and primary biliary cirrhosis),schistosomiasis, cholangitis (e.g., sclerosing cholangitis), andautoimmune-induced hepatitis), kidney fibrosis (e.g., tubulointerstitialfibrosis, scleroderma, diabetic nephritis, and glomerular nephritis),dermal fibrosis (e.g., scleroderma, hypertrophic and keloid scarring,nephrogenic fibrosing dermatopathy, and burns), myelofibrosis,neurofibromatosis, fibroma, intestinal fibrosis, and fibrotic adhesionsresulting from surgical procedures), heart fibrosis (e.g., fibrosisassociated with myocardial infarction), vascular fibrosis (e.g.,fibrosis associated with postangioplasty arterial restenosis andatherosclerosis), eye fibrosis (e.g., fibrosis associated withpost-cataract surgery, proliferative vitreoretinopathy, andretro-orbital fibrosis), and bone marrow fibrosis (e.g., idiopathicmyelofibrosis and drug-induced myelofibrosis). The fibrosis can beorgan-specific or systemic (e.g., systemic sclerosis and fibrosisassociated with GVHD). In some embodiments, the fibrotic disorder ispulmonary fibrosis. In some embodiments, the pulmonary fibrosis isfibrosing interstitial pneumonia. In some embodiments, the pulmonaryfibrosis is idiopathic pulmonary fibrosis (IPF), also known ascryptogenic fibrosing alveolitis. In some embodiments, the IPF isgender, age and physiology (GAP)-stage I. In some embodiments, the IPFis GAP-stage II. In some embodiments, the IPF is GAP-stage III. In someembodiments, the pulmonary fibrosis is sporadic IPF. In someembodiments, the pulmonary fibrosis is familial pulmonary fibrosis. Insome embodiments, the pulmonary fibrosis is combined pulmonary fibrosisand emphysema. In some embodiments, the pulmonary fibrosis is associatedwith one or more of the following: usual interstitial pneumonia;idiopathic interstitial pneumonia; desquamative interstitial pneumonia;respiratory bronchiolitis-interstitial lung disease; acute interstitialpneumonia; nonspecific interstitial pneumonia; sarcoidosis; cryptogenicorganizing pneumonia; eosinophilic pneumonia; infection; exposure tooccupational or environmental agents; cigarette smoking; interstitiallung disease induced by drugs or radiation; rheumatic disease-associatedinterstitial lung disease; lymphoid interstitial pneumonia;pleuropulmonary fibroelastosis; pulmonary Langerhans cell histiocytosis;systemic sclerosis-interstitial lung disease; Hermansky-Pudlak syndrome;and telomeropathy.

In some embodiments, the pulmonary disorder, autoimmune disorder,inflammatory disorder, fibrotic disorder, neutrophilic disorder, oreosinophilic disorder is chronic obstructive pulmonary disease (COPD).In some embodiments, the COPD is Global Initiative for ChronicObstructive Lung Disease (GOLD) category A. In some embodiments, theCOPD is GOLD category B. In some embodiments, the COPD is GOLD categoryC. In some embodiments, the COPD is GOLD category D. In someembodiments, the COPD is chronic bronchitis. In some embodiments, theCOPD is emphysema. In some embodiments, the emphysema is proximalacinar, panacinar, or distal acinar emphysema. In some embodiments, theemphysema is cigarette-induced emphysema. In some embodiments, the COPDis associated with exposure to particulate dusts, chemical fumes, and/orair pollution. In some embodiments, the COPD is associated with impairedlung development. In some embodiments, the COPD is chronic obstructiveasthma. In some embodiments, the COPD is associated with alpha-1antitrypsin deficiency. In some embodiments, the COPD is associated withserine protease inhibitor clade E, member 2 (SERPINE2) disruption. Insome embodiments, the COPD is COPD with persistent systemicinflammation. In some embodiments, the COPD is eosinophilic or T-helpertype 2 (T_(H)2) high COPD. In some embodiments, the COPD is COPD withpersistent bacterial colonization. In some embodiments, the COPD is COPDwith frequent exacerbations. In some embodiments, the autoimmunedisorder, inflammatory disorder, fibrotic disorder, neutrophilicdisorder, or eosinophilic disorder is asthma-COPD overlap syndrome(ACOS). In some embodiments, the ACOS is eosinophil-predominant,neutrophil-predominant, mixed-pattern, or no inflammation(paucigranulocytic) ACOS. In some embodiments, the autoimmune disorder,inflammatory disorder, fibrotic disorder, a neutrophilic disorder, or aneosinophilic disorder is COPD-obstructive sleep apnea (OSA) overlapsyndrome.

The above lists are not all-inclusive, and it will be understood by theskilled artisan that a disease or disorder may fall within variouscategories.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The terms “pharmaceutical formulation” and “pharmaceutical composition”are used interchangeably herein, and refer to a preparation which is insuch form as to permit the biological activity of an active ingredientcontained therein to be effective, and which contains no additionalcomponents which are unacceptably toxic to a subject to which theformulation would be administered. Such formulations are sterile. In apreferred embodiment, the pharmaceutical composition or pharmaceuticalformulation is administered to a human subject.

A “sterile” pharmaceutical formulation is aseptic or free or essentiallyfree from all living microorganisms and their spores.

A “stable” pharmaceutical formulation is one in which the protein (e.g.,an antibody, such as an anti-tryptase antibody) therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected amount of light exposure and/ortemperature for a selected time period. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways,including evaluation of aggregate formation (for example, using sizeexclusion chromatography, by measuring turbidity, and/or by visualinspection); evaluation of ROS formation (for example, by using a lightstress assay or an AAPH stress assay); oxidation of specific amino acidresidues of the protein (for example, a Trp residue and/or a Met residueof a monoclonal antibody); by assessing charge heterogeneity usingcation exchange chromatography, image capillary isoelectric focusing(icIEF) or capillary zone electrophoresis; amino-terminal orcarboxy-terminal sequence analysis; mass spectrometric analysis;SDS-PAGE analysis to compare reduced and intact antibody; peptide map(for example, tryptic or LYS-C) analysis; evaluating biological activityor target binding function of the protein (e.g., antigen bindingfunction of an antibody); and the like. Instability may involve any oneor more of: aggregation, deamidation (e.g., Asn deamidation), oxidation(e.g., Met oxidation and/or Trp oxidation), isomerization (e.g., Aspisomerization), clipping/hydrolysis/fragmentation (e.g., hinge regionfragmentation), succinimide formation, unpaired cysteine(s), N-terminalextension, C-terminal processing, glycosylation differences, and thelike.

An antibody (e.g., an anti-tryptase antibody) “retains its physicalstability” in a pharmaceutical formulation if it shows no signs or verylittle of aggregation, precipitation, fragmentation, and/or denaturationupon visual examination of color and/or clarity, or as measured by UVlight scattering or by size exclusion chromatography.

An antibody (e.g., an anti-tryptase antibody) “retains its chemicalstability” in a pharmaceutical formulation, if the chemical stability ata given time is such that the antibody is considered to still retain itsbiological activity as defined below. Chemical stability can be assessedby detecting and quantifying chemically altered forms of the antibody.Chemical alteration may involve protein oxidation which can be evaluatedusing tryptic peptide mapping, reverse-phase high-performance liquidchromatography (HPLC) and liquid chromatography-mass spectrometry(LC/MS), for example. Other types of chemical alteration include chargealteration of the antibody which can be evaluated by ion-exchangechromatography or icIEF, for example.

An antibody (e.g., an anti-tryptase antibody) “retains its biologicalactivity” in a pharmaceutical formulation, if the biological activity ofthe antibody at a given time is within about 20% (such as within about10%) of the biological activity exhibited at the time the pharmaceuticalformulation was prepared (within the errors of the assay), as determinedfor example in an antigen binding assay or an in vitro inhibitory assayfor a monoclonal antibody (e.g., an anti-tryptase monoclonal antibody).In some embodiments, the biological activity of an antibody at a giventime is within about 25%, about 30%, about 35%, about 40%, about 45%,about 50% of the biological activity exhibited at the time thepharmaceutical formulation was prepared.

As used herein, “biological activity” of an antibody (e.g., ananti-tryptase antibody) refers to the ability of the antibody to bindits target, for example the ability of a monoclonal antibody to bind toan antigen. It can further include a biological response which can bemeasured in vitro or in vivo. Such activity may be antagonistic oragonistic.

A protein (e.g., an antibody, such as an anti-tryptase antibody) whichis “susceptible to oxidation” is one comprising one or more residue(s)that has been found to be prone to oxidation such as, but not limitedto, methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp),and tyrosine (Tyr). For example, a tryptophan amino acid in the Fabportion of a monoclonal antibody or a methionine amino acid in the Fcportion of a monoclonal antibody may be susceptible to oxidation.

The term “percent oxidation” refers to the percentage of antibodies in aformulation (e.g., a pharmaceutical composition) that are oxidized at aparticular amino acid residue, for example, a Trp residue (e.g., Trp100in HVR-H3 of hu31A.v11) or a Met residue. Percent oxidation can bedetermined by, e.g., mass spectrometry (MS) analysis of one or moretryptic peptides, in which one or more particular oxidation-prone aminoacid residues reside. In certain embodiments, the percentage oxidationof Trp100 in HVR-H3 of hu31A.v11 is determined by the mass of oxidizedtryptic peptide in which Trp 100 resides, over the mass of the overall(oxidized and non-oxidized) tryptic peptide, as determined by MSanalysis. Percent oxidation may be determined, for example, within 9months, 12 months, 18 months, or two years from the initial productionof an antibody or pharmaceutical composition thereof.

The term “is determined following an AAPH stress test,” as used herein,means that the percent oxidation at a particular amino acid residue (forexample, at Trp100 in HVR-H3 of hu31A.v11) is determined by massspectrometry analysis of tryptic peptides following formulating theantibody at 150 mg/ml in 5 mM AAPH for 25 h at 40° C., for example, asdescribed in Example 5. The stressed antibody is digested with trypsinand the digested peptides were subjected to ultra high performanceliquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) todetermine the percentage of oxidation.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention preferably has a pH in the range from about 4.5to about 8.0 (e.g., about 4.5, about 5, about 5.5, about 6, about 6.5,about 7, about 7.5, or about 8), e.g., about pH 5.5. For example,histidine acetate is an example of a buffer that will control the pH inthis range. Another suitable buffer is arginine succinate and/orhistidine succinate.

A “preservative” is a compound which can be optionally included in theformulation to essentially reduce bacterial action therein, thusfacilitating the production of a multi-use formulation, for example.Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkylgroups are long-chain compounds), and benzethonium chloride. Other typesof preservatives include aromatic alcohols such as phenol, butyl, andbenzyl alcohol; alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol. In oneembodiment, the preservative herein is benzyl alcohol.

As used herein, a “surfactant” refers to a surface-active agent,preferably a nonionic surfactant. Examples of surfactants herein includepolysorbate (for example, polysorbate 20 and polysorbate 80); poloxamer(e.g., poloxamer 188); TRITON®; sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONIC® type block copolymers,e.g., PLURONIC® F-68); and the like. In one embodiment, the surfactantherein is polysorbate 20. In yet another embodiment, the surfactantherein is poloxamer 188.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al. “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, HumanaPress (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,3-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows, and sheep), sport animals, pets (such as cats, dogs and horses),primates (e.g., humans and non-human primates such as monkeys (e.g.,cynomolgus monkeys)), and rodents (e.g., mice and rats).

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-tryptase antibody of the invention or anadditional therapeutic agent) or a composition (e.g., a pharmaceuticalcomposition, e.g., a pharmaceutical composition including ananti-tryptase antibody of the invention, optionally also including anadditional therapeutic agent, which may include an excipient such as anantioxidant (e.g., N-acetyltryptophan and/or methionine)) to a subject.The compositions utilized in the methods described herein can beadministered, for example, intravitreally, intramuscularly,intravenously, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intrathecally,intranasally, intravaginally, intrarectally, topically, intratumorally,peritoneally, subcutaneously, subconjunctivally, intravesicularly,mucosally, intrapericardially, intraumbilically, intraocularly,intraorbitally, orally, topically, transdermally, periocularly,conjunctivally, subtenonly, intracamerally, subretinally, retrobulbarly,intracanalicularly, by inhalation, by injection, by implantation, byinfusion, by continuous infusion, by localized perfusion bathing targetcells directly, by catheter, by lavage, in cremes, or in lipidcompositions. The compositions utilized in the methods described hereincan also be administered systemically or locally. The method ofadministration can vary depending on various factors (e.g., the compoundor composition being administered and the severity of the condition,disease, or disorder being treated).

An “effective amount” or “therapeutically effective amount” of an agent,e.g., an anti-tryptase antibody or a pharmaceutical formulation (e.g., apharmaceutical formulation that includes an anti-tryptase antibody,which may include an excipient such as an antioxidant (e.g.,N-acetyltryptophan and/or methionine)), refers to an amount effective,at dosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result. The therapeutically effective amountof the antibody or antibody fragment (e.g., an anti-tryptase antibody),or composition thereof, may ameliorate or treat the disorder or disease,or prevent, reduce, ameliorate, or treat symptoms associated with thedisorder or disease.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease. A patient may be successfully “treated” forasthma if, for example, after receiving an asthma therapy, the patientshows observable and/or measurable reduction in or absence of one ormore of the following: recurrent wheezing, coughing, trouble breathing,chest tightness, symptoms that occur or worsen at night, symptoms thatare triggered by cold air, exercise or exposure to allergens.

The term “interleukin-5 (IL-5),” as used herein, refers to any nativeIL-5 from any vertebrate source, including mammals such as primates(e.g., humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed IL-5, matureIL-5, as well as any form of IL-5 that results from post-translationalmodifications. The term also encompasses naturally occurring variants ofIL-5, such as splice variants or allelic variants. The amino acidsequence of an exemplary IL-5 can be found, for example, under UniProtKBaccession number P05113.

The term “IL-5 axis binding antagonist” refers to a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of IL-5 with one or more ofits binding partners, such as IL-5 receptor, alpha (IL5RA). ExemplaryIL-5 axis binding antagonists that can be used in the methods of theinvention include, for example, IL-5 binding antagonists (e.g.,anti-IL-5 antibodies (e.g., mepolizumab, benralizumab, and reslizumab)and IL-5 receptor binding antagonists (e.g., anti-IL-5R antibodies)).

As used herein, “interleukin-13 (IL-13)” refers to any native IL-13 fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats), unless otherwise indicated. IL-13 isa cytokine secreted by many cell types, including T helper type 2 (Th2)cells. The term encompasses “full-length,” unprocessed IL-13, matureIL-13, as well as any form of IL-13 that results from post-translationalmodifications. The amino acid sequence of an exemplary human IL-13 canbe found, for example, under UniProtKB accession number P35225.

The term “IL-13 axis binding antagonist” refers to a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of IL-13 with one or more ofits binding partners, such as IL-4 receptor alpha (IL4Rα), IL-13receptor alpha1 (IL13RA1) and IL-13 receptor alpha2 (IL13RA2). IL-13axis binding antagonists include IL-13 binding antagonists (e.g.,anti-IL-13 antibodies, for example, lebrikizumab, 228B/C-1, 228A-4,227-26, and 227-43 (see, for example, U.S. Pat. Nos. 7,674,459;8,067,199; 8,088,618; 8,318,160; and 8,734,797) and IL-13 receptorbinding antagonists (e.g., an anti-IL4Rα antibody, an anti-IL13RA1antibody, or an anti-IL13RA2 antibody).

As used herein, “interleukin-17 (IL-17)” refers to any native IL-17 fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats), unless otherwise indicated, andincludes family members IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, andIL-17F. The term encompasses “full-length,” unprocessed IL-17, matureIL-17, as well as any form of IL-17 that results from post-translationalmodifications. The amino acid sequence of an exemplary human IL-17A canbe found, for example, under UniProtKB accession number Q16552. Theamino acid sequence of an exemplary human IL-17B can be found, forexample, under UniProtKB accession number Q9UHF5. The amino acidsequence of an exemplary human IL-17C can be found, for example, underUniProtKB accession number Q9P0M4. The amino acid sequence of anexemplary human IL-17D can be found, for example, under UniProtKBaccession number Q8TAD2. The amino acid sequence of an exemplary humanIL-17E can be found, for example, under UniProtKB accession numberQ9H293. The amino acid sequence of an exemplary human IL-17F can befound, for example, under UniProtKB accession number Q96PD4.

The term “IL-17 axis binding antagonist” refers to a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of IL-17 with one or more ofits binding partners, such as interleukin-17 receptor (IL-17R) familymember proteins interleukin 17 receptor A (IL17RA), interleukin 17receptor B (IL17RB), interleukin 17 receptor C (IL17RC), interleukin 17receptor D (IL17RD), interleukin 17 receptor E (IL17RE), and interleukin17 receptor E-like (IL7REL). Exemplary IL-17 axis binding antagonistsinclude, for example, IL-17 binding antagonists (e.g., anti-IL-17antibodies (e.g., secukinumab (AIN417), ixekizumab (LY2439821),bimekizumab, and NI-1401) and IL-17 receptor binding antagonists (e.g.,anti-IL-17R antibodies (e.g., brodalumab (AMG-827))). See, e.g., WO2006/013107, WO 2007/070750, WO 2012/156219, and U.S. Pat. No.8,715,669.

The term “interleukin-33 (IL-33),” as used herein, refers to any nativeIL-33 from any vertebrate source, including mammals such as primates(e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats),unless otherwise indicated. IL-33 is also referred to in the art asnuclear factor of high endothelial venules (NF-HEV; see, e.g.,Baekkevold et al. Am. J. Pathol. 163(1): 69-79, 2003), DVS27, C9orf26,and interleukin-1 family member 11 (IL-1F11). The term encompasses“full-length,” unprocessed IL-33, mature IL-33, as well as any form ofIL-33 that results from post-translational modifications. Humanfull-length, unprocessed IL-33 contains 270 amino acids (a.a.) and mayalso be referred to as IL-33₁₋₂₇₀. Processed forms of human IL-33include, for example, IL-33₉₅₋₂₇₀, IL-33₉₉₋₂₇₀, IL-33₁₀₉₋₂₇₀,IL-33₁₁₂₋₂₇₀, IL-33₁₋₁₇₈, and IL-33₁₇₉₋₂₇₀ (Lefrangais et al. Proc.Natl. Acad. Sci. 109(5):1673-1678, 2012 and Martin, Semin. Immunol. 25:449-457, 2013). In some embodiments, processed forms of human IL-33,e.g., IL-33₉₅₋₂₇₀, IL-33₉₉₋₂₇₀, IL-33₁₀₉₋₂₇₀, or other forms processedby proteases such as calpain, proteinase 3, neutrophil elastase, andcathepsin G may have increased biological activity compared tofull-length IL-33. The term also encompasses naturally occurringvariants of IL-33, for example, splice variants (e.g., theconstitutively active splice variant spIL-33 which lacks exon 3, Hong etal. J. Biol. Chem. 286(22):20078-20086, 2011) or allelic variants. IL-33may be present within a cell (e.g., within the nucleus) or as a secretedcytokine form. Full-length IL-33 protein contains a helix-turn-helixDNA-binding motif including nuclear localization sequence (a.a. 1-75 ofhuman IL-33), which includes a chromatin binding motif (a.a. 40-58 ofhuman IL-33). Forms of IL-33 that are processed and secreted lack theseN-terminal motifs. The amino acid sequence of an exemplary human IL-33can be found, for example, under UniProtKB accession number 095760.

The terms “interleukin 1 receptor-like 1 (IL1RL1)” and “ST2,” usedinterchangeably herein, refer to any native ST2 from any vertebratesource, including mammals such as primates (e.g., humans) and rodents(e.g., mice and rats), unless otherwise indicated. ST2 is also referredto in the art as DER4, T1, and FIT-1. The term encompasses“full-length,” unprocessed ST2, mature ST2, as well as any form of ST2that results from post-translational modifications. At least fourisoforms of ST2 are known in the art, including soluble (sST2, alsoknown as IL1RL1-a) and transmembrane (ST2L, also known as IL1RL1-b),which arise from differential mRNA expression from a dual promotersystem, and ST2V and ST2LV, which arise from alternative splicing, asdescribed below. The domain structure of ST2L includes threeextracellular immunoglobulin-like C2 domains, a transmembrane domain,and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. sST2 lacksthe transmembrane and cytoplasmic domains contained within ST2L andincludes a unique 9 amino acid (a.a.) C-terminal sequence (see, e.g.,Kakkar et al. Nat. Rev. Drug Disc. 7: 827-840, 2008). sST2 can functionas a decoy receptor to inhibit soluble IL-33. The term also encompassesnaturally occurring variants of ST2, e.g., splice variants (e.g., ST2V,which lacks the third immunoglobulin motif and has a unique hydrophobictail, and ST2LV, which lacks the transmembrane domain of ST2L) orallelic variants (e.g., variants that are protective against asthma riskor that confer asthma risk as described herein). The amino acid sequenceof an exemplary human ST2 can be found, for example, under UniProtKBaccession number Q01638. ST2 is a part of the IL-33 receptor along withthe co-receptor protein IL-1RAcP. Binding of IL-33 to ST2 and theco-receptor interleukin-1 receptor accessory protein (IL-1RAcP) forms a1:1:1 ternary signaling complex to promote downstream signaltransduction (see, e.g., Lingel et al. Structure 17(10): 1398-1410,2009, and Liu et al. Proc. Natl. Acad. Sci. 110(37): 14918-14924, 2013).

By “IL-33 axis” is meant a nucleic acid (e.g., a gene or mRNAtranscribed from the gene) or polypeptide that is involved in IL-33signal transduction. For example, the IL-33 axis may include the ligandIL-33, a receptor (e.g., ST2 and/or IL-1RAcP), adaptor molecules (e.g.,MyD88), or proteins that associate with receptor molecules and/oradaptor molecules (e.g., kinases, such as interleukin-1receptor-associated kinase 1 (IRAK1) and interleukin-1receptor-associated kinase 4 (IRAK4), or E3 ubiquitin ligases, such asTNF receptor associated factor 6 (TRAF6)).

An “IL-33 axis binding antagonist” refers to a molecule that inhibitsthe interaction of an IL-33 axis binding partner with one or more of itsbinding partners. As used herein, an IL-33 axis binding antagonistincludes IL-33 binding antagonists, ST2 binding antagonists, and IL1RAcPbinding antagonists. Exemplary IL-33 axis binding antagonists includeanti-IL-33 antibodies and antigen-binding fragments thereof (e.g.,anti-IL-33 antibodies such as ANB-020 (AnaptysBio, Inc.) or any of theantibodies described in EP1725261, U.S. Pat. No. 8,187,596,WO2011031600, WO2014164959, WO2015099175 or WO2015106080, which are eachincorporated herein by reference in their entirety); polypeptides thatbind IL-33 and/or its receptor (ST2 and/or IL-1RAcP) and blockligand-receptor interaction (e.g., ST2-Fc proteins, such as thosedescribed in WO 2014/152195, which is herein incorporated by referencein its entirety; immunoadhesins, peptibodies, and soluble ST2, orderivatives thereof); anti-IL-33 receptor antibodies (e.g., anti-ST2antibodies, for example, AMG-282 (Amgen) or STLM15 (Janssen) or any ofthe anti-ST2 antibodies described in WO 2013/173761 and WO 2013/165894,which are each incorporated herein by reference in their entirety; orST2-Fc proteins, such as those described in WO 2013/173761; WO2013/165894; or WO 2014/152195, which are each incorporated herein byreference in their entirety); and IL-33 receptor antagonists, such assmall molecule inhibitors, aptamers that bind IL-33, and nucleic acidsthat hybridize under stringent conditions to IL-33 axis nucleic acidsequences (e.g., short interfering RNAs (siRNA) or clustered regularlyinterspaced short palindromic repeat RNAs (CRISPR-RNA or crRNA),including single guide RNAs (sgRNAs) having a crRNA and tracrRNAsequence as described in Mali et al. (Science. 339: 823-26, 2013), whichis incorporated herein by reference in its entirety).

The terms “anti-IL-33 antibody,” an “antibody that binds to IL-33,” and“antibody that specifically binds IL-33” refer to an antibody that iscapable of binding IL-33 with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting IL-33.In one embodiment, the extent of binding of an anti-IL-33 antibody to anunrelated, non-IL-33 protein is less than about 10% of the binding ofthe antibody to IL-33 as measured, e.g., by a radioimmunoassay (RIA). Incertain embodiments, an antibody that binds to IL-33 has a dissociationconstant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g.,from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-IL-33 antibodybinds to an epitope of IL-33 that is conserved among IL-33 fromdifferent species.

The term “ST2 binding antagonist” refers to a molecule that inhibits theinteraction of an ST2 with IL-33, IL1RAcP, and/or a second ST2 molecule.The ST2 binding antagonist may be a protein, such as an “ST2-Fc protein”that includes an IL-33-binding domain (e.g., all or a portion of an ST2or IL1RAcP protein) and a multimerizing domain (e.g., an Fc portion ofan immunoglobulin, e.g., an Fc domain of an IgG selected from theisotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within eachisotype group), which are attached to one another either directly orindirectly through a linker (e.g., a serine-glycine (SG) linker,glycine-glycine (GG) linker, or variant thereof (e.g., a SGG, a GGS, anSGS, or a GSG linker)), and includes, but is not limited to, ST2-Fcproteins and variants thereof described in WO 2013/173761, WO2013/165894, and WO 2014/152195, which are each incorporated herein byreference in their entirety. In some embodiments, a ST2 bindingantagonist may be an anti-ST2 antibody, for example, AMG-282 (Amgen) orSTLM15 (Janssen) or any of the anti-ST2 antibodies described in WO2013/173761 and WO 2013/165894.

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Percent (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the polypeptide being compared, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN, orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The amino acid sequences described herein are contiguous amino acidsequences unless otherwise specified.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors” or “expression vectors”). In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably.

II. Compositions and Methods

In one aspect, the invention is based, in part, on novel antibodies thatbind to tryptase. In another aspect, the invention is based, in part, onthe discovery that particular residues (e.g., HVR residues, such asHVR-H3 W100, which refers to W100 of the VH domain of the anti-tryptaseantibody hu31A.v11) of anti-tryptase antibodies may be susceptible tooxidation. The invention provides pharmaceutical compositions thatinclude antioxidants (e.g., N-acetyltryptophan and/or methionine) toreduce or prevent oxidation of antibodies described herein (e.g.,anti-tryptase antibodies). Other suitable antioxidant excipientsinclude, without limitation, free tryptophan, cyclodextrins, Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), pyridoxine,polyols (e.g., mannitol), and metal chelators (e.g., EDTA). See, e.g.,Ji et al., Biotechnology 98:4485-4500, 2009. Antibodies andpharmaceutical compositions of the invention are useful, e.g., for thediagnosis and/or treatment of disorders (e.g., a pulmonary disorder, anautoimmune disorder, an inflammatory disorder, a fibrotic disorder, agranulocytic (neutrophilic or eosinophilic) disorder, a monocyticdisorder, a lymphocytic disorder, a disorder associated with increasednumbers or distribution of normal or aberrant tissue resident cells(such as mast cells, macrophages, or lymphocytes) or stromal cells (suchas fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia, or a tryptase-associated disorder or a tryptase-mediateddisorder. In another aspect, the invention provides lyophilizedpharmaceutical compositions to reduce or eliminate oxidation ofantibodies described herein (e.g., anti-tryptase antibodies).

A. Exemplary Anti-Tryptase Antibodies

The invention provides isolated antibodies that bind to tryptase. Incertain embodiments, an anti-tryptase antibody of the invention binds totryptase with a K_(D) of about 100 nM or lower (e.g., 100 nM or lower,10 nM or lower, 1 nM or lower, 100 pM or lower, 10 pM or lower, 1 pM orlower, or 0.1 pM or lower). In some embodiments, the antibody bindstryptase with a K_(D) of 10 nM or lower (e.g., 10 nM or lower, 1 nm orlower, 100 pM or lower, 10 pM or lower, 1 pM or lower, or 0.1 pM orlower). In some embodiments, the antibody binds tryptase with a K_(D) of1 nM or lower (e.g., 1 nm or lower, 100 pM or lower, 10 pM or lower, 1pM or lower, or 0.1 pM or lower). In some embodiments, the antibodybinds tryptase with a K_(D) of 0.5 nM or lower (e.g., 0.5 nm or lower,400 pM or lower, 300 pM or lower, 200 pM or lower, 100 pM or lower, 50pM or lower, 25 pM or lower, 10 pM or lower, 1 pM or lower, or 0.1 pM orlower). In some embodiments, the antibody binds tryptase with a K_(D)between about 0.1 nM to about 0.5 nM (e.g., about 0.1 nM, about 0.2 nM,about 0.3 nM, about 0.4 nM, or about 0.5 nM). In some embodiments, theantibody binds tryptase with a K_(D) between about 1 pM to about 500 pM,about 1 pM to about 400 pM, about 1 pM to about 300 pM, about 1 pM toabout 200 pM, about 1 pM to about 100 pM, about 1 pM to about 50 pM,about 25 pM to about 500 pM, about 25 pM to about 400 pM, about 25 pM toabout 300 pM, about 25 pM to about 100 pM, about 50 pM to about 500 pM,about 50 pM to about 450 pM, about 50 pM to about 425 pM, about 50 pM toabout 400 pM, about 50 pM to about 375 pM, about 50 pM to about 350 pM,about 50 pM to about 325 pM, about 50 pM to about 300 pM, about 50 pM toabout 275 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM,about 50 pM to about 180 pM, about 50 pM to about 175 pM, about 50 pM toabout 150 pM, about 50 pM to about 125 pM, about 50 pM to about 100 pM,about 50 pM to about 75 pM, about 100 pM to about 500 pM, about 100 pMto about 475 pM, about 100 pM to about 450 pM, about 100 pM to about 425pM, about 100 pM to about 400 pM, about 100 pM to about 375 pM, about100 pM to about 350 pM, about 100 pM to about 325 pM, about 100 pM toabout 300 pM, about 100 pM to about 275 pM, about 100 pM to about 250pM, about 100 pM to about 225 pM, about 100 pM to about 200 pM, about100 pM to about 180 pM, about 100 pM to about 175 pM, about 100 pM toabout 150 pM, about 100 pM to about 125 pM, about 150 pM to about 500pM, about 150 pM to about 475 pM, about 150 pM to about 450 pM, about150 pM to about 425 pM, about 150 pM to about 400 pM, about 150 pM toabout 375 pM, about 150 pM to about 350 pM, about 150 pM to about 325pM, about 150 pM to about 300 pM, about 150 pM to about 375 pM, about150 pM to about 350 pM, about 150 pM to about 325 pM, about 150 pM toabout 300 pM, about 150 pM to about 275 pM, about 150 pM to about 225pM, about 150 pM to about 200 pM, about 175 pM to about 500 pM, about175 pM to about 475 pM, about 175 pM to about 450 pM, about 175 pM toabout 425 pM, about 175 pM to about 400 pM, about 175 pM to about 375pM, about 175 pM to about 350 pM, about 175 pM to about 325 pM, about175 pM to about 300 pM, or about 180 pM to about 400 pM. In someembodiments, the antibody binds tryptase with a K_(D) of about 0.4 nM.In some embodiments, the antibody binds tryptase with a K_(D) of about0.2 nM. In some embodiments, the antibody binds tryptase with a K_(D) ofabout 0.18 nM. In some embodiments, the tryptase is human tryptase, forexample, human tryptase beta (e.g., human tryptase beta 1, humantryptase beta 2, and/or human tryptase beta 3). In some embodiments, theK_(D) is determined in a BIACORE® SPR assay. In certain embodiments, thetryptase is human tryptase alpha. In certain embodiments, the antibodyis a human or humanized antibody.

In another example, in some embodiments, an anti-tryptase antibody ofthe invention (including any of the preceding anti-tryptase antibodies)is capable of inhibiting an activity of tryptase. In some embodiments,an anti-tryptase antibody of the invention is capable of inhibiting aproteolytic activity of tryptase, for example, as determined in an invitro tryptase enzymatic assay. In some embodiments, an artificialsubstrate, for example, the synthetic peptide S-2288 can be used as asubstrate in an in vitro tryptase enzymatic assay. In some embodiments,an anti-tryptase antibody of the invention is capable of inhibiting theactivity of human tryptase with a half-maximal inhibitory concentration(IC50) of about 100 nM or lower (e.g., 100 nM or lower, 10 nM or lower,5 nM or lower, 2.5 nM or lower, 1 nM or lower, 100 pM or lower, 10 pM orlower, 1 pM or lower, or 0.1 pM or lower) as determined by an in vitrotryptase enzymatic assay, for example, using S-2288 as a substrate. Insome embodiments, the antibody is capable of inhibiting the activity ofhuman tryptase with an IC50 of about 10 nM or lower (e.g., 10 nM orlower, 5 nM or lower, 2.5 nM or lower, 1 nM or lower, 100 pM or lower,10 pM or lower, 1 pM or lower, or 0.1 pM or lower) as determined by anin vitro tryptase enzymatic assay, for example, using S-2288 as asubstrate. In some embodiments, the antibody is capable of inhibitingthe activity of human tryptase with an IC50 of about 2.5 nM or lower(e.g., 2.5 nM or lower, 1 nM or lower, 100 pM or lower, 10 pM or lower,1 pM or lower, or 0.1 pM or lower) as determined by an in vitro tryptaseenzymatic assay, for example, using S-2288 as a substrate. In someembodiments, the antibody is capable of inhibiting the activity of humantryptase with an IC50 of about 0.1 nM to about 2 nM (e.g., about 0.1 nM,about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM,about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1.0 nM, about 1.1 nM,about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM,about 1.7 nM, about 1.8 nM, about 1.9 nM, or about 2.0 nM). In someembodiments, the antibody is capable of inhibiting the activity of humantryptase with an IC50 of about 0.5 nM to about 2.5 nM (e.g., about 0.5nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1.0nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, or about 2.5nM). In some embodiments, the antibody is capable of inhibiting theactivity of human tryptase with an IC50 of about 1 pM to about 2.5 nM,about 25 pM to about 2.5 nM, about 50 pM to about 2.5 nM, about 75 pM toabout 2.5 nM, about 100 pM to about 2.5 nM, about 125 pM to about 2.5nM, about 150 pM to about 2.5 nM, about 175 pM to about 2.5 nM, about200 pM to about 2.5 nM, about 225 pM to about 2.5 nM, about 250 pM toabout 2.5 nM, about 300 pM to about 2.5 nM, about 325 pM to about 2.5nM, about 325 pM to about 2.5 nM, about 350 pM to about 2.5 nM, about375 pM to about 2.5 nM, about 400 pM to about 2.5 nM, about 425 pM toabout 2.5 nM, about 450 pM to about 2.5 nM, about 500 pM to about 2.5nM, about 450 pM to about 2.5 nM, about 500 pM to about 2.5 nM, about550 pM to about 2.5 nM, about 600 pM to about 2.5 nM, about 650 pM toabout 2.5 nM, about 700 pM to about 2.5 nM, about 750 pM to about 2.5nM, about 800 pM to about 2.5 nM, about 850 pM to about 2.5 nM, about900 pM to about 2.5 nM, about 950 pM to about 2.5 nM, about 1 nM toabout 2.5 nM, about 1.1 nM to about 2.5 nM, about 1.2 nM to about 2.5nM, about 1.3 nM to about 2.5 nM, about 1.4 nM to about 2.5 nM, about1.5 nM to about 2.5 nM, about 1.6 nM to about 2.5 nM, about 1.7 nM toabout 2.5 nM, about 1.8 nM to about 2.5 nM, about 1.9 nM to about 2.5nM, about 2.0 nM to about 2.5 nM, about 2.1 nM to about 2.5 nM, about2.2 nM to about 2.5 nM, about 2.3 nM to about 2.5 nM, about 500 pM toabout 1.9 pM, about 750 pM to about 1.9 pM, about 1 nM to about 1.9 pM,about 1.25 nM to about 1.9 pM, about 1.5 nM to about 1.9 pM, about 1 nMto about 1.85 nM, about 1.25 nM to about 1.85 nM, about 1.25 nM to about1.85 nM, about 1.5 nM to about 1.85 nM, about 1 nM to about 1.8 nM,about 1.25 nM to about 1.8 nM, about 1.5 nM to about 1.8 nM, or about1.6 nM to about 1.8 nM. In some embodiments, the antibody is capable ofinhibiting the activity of human tryptase with an IC50 of about 1.8 nM.In other embodiments, the antibody is capable of inhibiting the activityof human tryptase with an IC50 of about 0.5 nM to about 1 nM (e.g.,about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, orabout 1.0 nM). In some embodiments, the antibody is capable ofinhibiting the activity of human tryptase with an IC50 of about 1 pM toabout 1 nM, about 25 pM to about 1 nM, about 50 pM to about 1 nM, about75 pM to about 1 nM, about 100 pM to about 1 nM, about 125 pM to about 1nM, about 150 pM to about 1 nM, about 175 pM to about 1 nM, about 200 pMto about 1 nM, about 225 pM to about 1 nM, about 250 pM to about 1 nM,about 300 pM to about 1 nM, about 325 pM to about 1 nM, about 350 pM toabout 1 nM, about 375 pM to about 1 nM, about 400 pM to about 1 nM,about 425 pM to about 1 nM, about 450 pM to about 1 nM, about 500 pM toabout 1 nM, about 450 pM to about 1 nM, about 500 pM to about 1 nM,about 550 pM to about 1 nM, about 600 pM to about 1 nM, about 650 pM toabout 1 nM, about 700 pM to about 1 nM, about 750 pM, about 250 pM toabout 800 pM, about 300 pM to about 800 pM, about 325 pM to about 800pM, about 325 pM to about 800 pM, about 350 pM to about 800 pM, about375 pM to about 800 pM, about 400 pM to about 800 pM, about 425 pM toabout 800 pM, about 450 pM to about 800 pM, about 500 pM to about 800pM, about 450 pM to about 800 pM, about 500 pM to about 800 pM, about550 pM to about 800 pM, about 600 pM to about 800 pM, about 650 pM toabout 800 pM, about 700 pM to about 800 pM, about 750 pM to about 800pM, about 1 pM to about 600 pM, about 25 pM to about 600 pM, about 50 pMto about 600 pM, about 75 pM to about 600 pM, about 100 pM to about 600pM, about 125 pM to about 600 pM, about 150 pM to about 600 pM, about175 pM to about 600 pM, about 200 pM to about 600 pM, about 225 pM toabout 600 pM, about 250 pM to about 600 pM, about 300 pM to about 600pM, about 325 pM to about 600 pM, about 325 pM to about 600 pM, about350 pM to about 600 pM, about 375 pM to about 600 pM, about 400 pM toabout 600 pM, about 425 pM to about 600 pM, about 450 pM to about 600pM, about 500 pM to about 600 pM, about 450 pM to about 600 pM, about500 pM to about 600 pM, or about 550 pM to about 600 pM. In someembodiments, the antibody is capable of inhibiting the activity of humantryptase with an IC50 of about 0.6 nM. In some embodiments, the tryptaseis human tryptase, for example, human tryptase beta (e.g., humantryptase beta 1, human tryptase beta 2, and/or human tryptase beta 3).In some instances, the inhibitory activity of the antibody is determineddescribed herein, for example, in the Examples (e.g., Example 1,particularly Section (A)(viii)(a)), or by other approaches known in theart. In certain embodiments, the antibody is a human or a humanizedantibody. In some embodiments, the antibody is capable of inhibiting theactivity of human tryptase as a monovalent antibody or antigen-bindingantibody fragment thereof (e.g., an Fab). In other embodiments, theantibody is capable of inhibiting the activity of human tryptase as abivalent antibody (e.g., an IgG antibody (e.g., an IgG1 or IgG4antibody) or an F(ab′)2).

In some instances, any of the anti-tryptase antibodies described hereincan inhibit tryptase-stimulated contraction of human primary airwaysmooth muscle cells. In other instances, any of the anti-tryptaseantibodies described herein can inhibit tryptase-stimulated contractionof human primary airway smooth muscle cells. In yet other instances, anyof the anti-tryptase antibodies described herein can inhibit tryptase orIgE-stimulated mast cell degranulation and/or histamine release. Infurther instances, any of the anti-tryptase antibodies described hereincan reduce the amount of active tryptase (e.g., in a sample such asbronchoalveolar lavage fluid or a nasosorption sample), for example,upon administration to a subject. For example, any of the anti-tryptaseantibodies described herein can reduce the amount of active tryptase byabout 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, or more. The reduction may be relative to a referenceamount of active tryptase, for example, the amount of active tryptase ina sample prior to administration of the anti-tryptase antibody.

In some instances, the antibody (e.g., the anti-tryptase antibody) mayinclude at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from: (a) an HVR-H1 comprising the amino acidsequence of X₁X₂GMX₃ (SEQ ID NO: 1), wherein X₁ is Asp or Ser, X₂ is Tyror Phe, and X₃ is Val or His; (b) an HVR-H2 comprising the amino acidsequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H₃ comprisingthe amino acid sequence of RX₁X₂X₃DWYFDV (SEQ ID NO: 3), wherein X₁ isAsn or Asp, X₂ is Tyr or Asn, and X₃ is Asp or Tyr; (d) an HVR-L1comprising the amino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) anHVR-L2 comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and(f) an HVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ IDNO: 6), or a combination of one or more of the above HVRs and one ormore variants thereof having at least about 80% sequence identity (e.g.,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1-6.

For instance, in some embodiments, the antibody (e.g., the anti-tryptaseantibody) may include at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from: (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6), or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 2 or 4-8.

In one particular example, in some embodiments, the antibody (e.g., theanti-tryptase antibody) may include (a) an HVR-H1 comprising the aminoacid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprising theamino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) anHVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ ID NO: 4);(e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQ ID NO:5); and (f) an HVR-L3 comprising the amino acid sequence of QHYHSYPLT(SEQ ID NO: 6). In some embodiments, the antibody (e.g., theanti-tryptase antibody) includes (a) a heavy chain variable (VH) domaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of SEQ ID NO: 9;(b) a light chain variable (VL) domain comprising an amino acid sequencehaving at least 90% identity to (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity), or the sequence of, the aminoacid sequence of SEQ ID NO: 10; or (c) a VH domain as in (a) and a VLdomain as in (b). In some embodiments, the antibody (e.g., theanti-tryptase antibody) includes one, two, three, or four of thefollowing heavy chain framework regions (FRs): (a) an FR-H1 comprisingthe amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:11); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 12); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR (SEQ ID NO: 13); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). Insome embodiments, the antibody (e.g., the anti-tryptase antibody)includes one, two, three, or four of the following light chain FRs: (a)an FR-L1 comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO: 15); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKSPKPWIY (SEQ ID NO: 16); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17); and (d) anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 18).In some embodiments, the antibody (e.g., the anti-tryptase antibody)includes a VH domain comprising the amino acid sequence of SEQ ID NO: 9and a VL domain comprising the amino acid sequence of SEQ ID NO: 10,such as the antibody hu31A.v11.

In another particular example, in some embodiments, the antibody (e.g.,the anti-tryptase antibody) may include (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RDNYDWYFDV (SEQ ID NO: 29);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6). In some embodiments, the antibody (e.g., theanti-tryptase antibody) includes (a) a heavy chain variable (VH) domaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of SEQ ID NO: 19;(b) a light chain variable (VL) domain comprising an amino acid sequencehaving at least 90% identity to (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity), or the sequence of, the aminoacid sequence of SEQ ID NO: 20; or (c) a VH domain as in (a) and a VLdomain as in (b). In some embodiments, the antibody (e.g., theanti-tryptase antibody) includes one, two, three, or four of thefollowing heavy chain framework regions (FRs): (a) an FR-H1 comprisingthe amino acid sequence of EVKLVESGGGSVQPGGSRKLSCAASGFTFS (SEQ ID NO:21); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 22); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNPKNTLFLQMSSLRSEDTAMYYCAR (SEQ ID NO: 23); and (d) an FR-H4comprising the amino acid sequence of WGTGTTVTVSS (SEQ ID NO: 24). Insome embodiments, the antibody (e.g., the anti-tryptase antibody)includes one, two, three, or four of the following light chain FRs: (a)an FR-L1 comprising the amino acid sequence of QIVLTQSPAIMSASPGEKVTISC(SEQ ID NO: 25); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGSSPKPWIY (SEQ ID NO: 26); (c) an FR-L3 comprising the amino acidsequence of GVPARFSGSGSGTSYSLTISSMEAEDAATYYC (SEQ ID NO: 27); and (d) anFR-L4 comprising the amino acid sequence of FGAGTKLELK (SEQ ID NO: 28).In some embodiments, the antibody (e.g., the anti-tryptase antibody)includes a VH domain comprising the amino acid sequence of SEQ ID NO: 19and a VL domain comprising the amino acid sequence of SEQ ID NO: 20,such as the antibody 31a.

In some instances, the antibody (e.g., the anti-tryptase antibody)includes (a) a heavy chain variable (VH) domain comprising an aminosequence having at least 90% sequence identity to (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or thesequence of, the amino acid sequence of any one of SEQ ID NOs: 9, 101,102, 103, and 104; (b) a light chain variable (V_(L)) domain comprisingan amino acid sequence having at least 90% identity to (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), orthe sequence of, the amino acid sequence of any one of SEQ ID NOs: 10,105, and 106; or (c) a VH domain as in (a) and a VL domain as in (b).For example, in some instances, the antibody comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 98 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 102. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 98 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 98 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 103. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 99 and a VL domain comprising the amino acidsequence of SEQ ID NO: 102. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 99 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 99 and a VL domain comprising the amino acidsequence of SEQ ID NO: 103. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 100 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 102. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 100 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 100 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 103. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 102. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 103. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 101 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 102. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 101 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 101 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 103.

In some instances, any of the preceding antibodies binds to an epitopeon human tryptase beta 1 comprising at least one, at least two, at leastthree, or all four residues selected from the group consisting of His51,Val80, Lys81, and Asp82 of SEQ ID NO: 71. In some embodiments, theantibody binds to an epitope on human tryptase beta 1 comprising atleast one, at least two, at least three, or all four residues selectedfrom the group consisting of His51, Val80, Lys81, and Asp82 of SEQ IDNO: 71. In some embodiments, the antibody binds to an epitope on humantryptase beta 1 comprising His51 and at least one, at least two, or allthree residues selected from the group consisting of Val 80, Lys81, andAsp82 of SEQ ID NO: 71. In some embodiments, the epitope on humantryptase beta 1 further comprises one or more amino acid residuesselected from the group consisting of Gln67, Leu83, Ala84, Ala85, Arg87,Pro103, Va104, Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71. In some embodiments, the epitope on human tryptase beta 1 comprisesat least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, or all twelve amino acid residues selected from the groupconsisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103, Val104, Ser105,Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71. In someembodiments, the epitope on human tryptase beta 1 comprises His51,Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Va104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is determined by an X-raycrystallography model. In some embodiments, the antibody is capable ofdissociating both the small interface of tetrameric human tryptase beta1 and the large interface of tetrameric human tryptase beta 1.

In some instances, any of the preceding anti-tryptase antibodiesincludes a paratope that binds tryptase (e.g., human tryptase beta 1)that includes one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid residues)selected from the group consisting of light chain variable region aminoacid residues Va30; Thr31; Tyr32; Tyr34; Arg50; Tyr90; His92; Ser93; andTyr94 and the heavy chain variable region amino acid residues Phe50;Ser52; Gly53; Ser54; Ser55; Thr56; Tyr58; Arg95; Tyr97; and Asp98.

For example, in some instances, the anti-tryptase antibody includes aparatope that binds tryptase (e.g., human tryptase beta 1) that includeslight chain variable region amino acid residues Va30, Thr31, Tyr32,Tyr34, Arg50, Tyr90, His92, Ser93, and Tyr94 or the heavy chain variableregion amino acid residues Phe50, Ser52, Gly53, Ser54, Ser55, Thr56,Tyr58, Arg95, Tyr97, and Asp98. In some instances, the anti-tryptaseantibody includes a paratope that binds tryptase (e.g., human tryptasebeta 1) that includes light chain variable region amino acid residuesVal30, Thr31, Tyr32, Tyr34, Arg50, Tyr90, His92, Ser93, and Tyr94 andthe heavy chain variable region amino acid residues Phe50, Ser52, Gly53,Ser54, Ser55, Thr56, Tyr58, Arg95, Tyr97, and Asp98.

In some instances, the antibody (e.g., the anti-tryptase antibody) mayinclude at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from: (a) an HVR-H1 comprising the amino acidsequence of GYAIT (SEQ ID NO: 30); (b) an HVR-H2 comprising the aminoacid sequence of GISSAATTFYSSWAKS (SEQ ID NO: 31); (c) an HVR-H3comprising the amino acid sequence of DPRGYGAALDRLDL (SEQ ID NO: 32);(d) an HVR-L1 comprising the amino acid sequence of QSIKSVYNNRLG (SEQ IDNO: 33); (e) an HVR-L2 comprising the amino acid sequence of ETSILTS(SEQ ID NO: 34); and (f) an HVR-L3 comprising the amino acid sequence ofAGGFDRSGDTT (SEQ ID NO: 35), or a combination of one or more of theabove HVRs and one or more variants thereof having at least about 80%sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof SEQ ID NOs: 30-35.

In some instances, the antibody (e.g., the anti-tryptase antibody)includes (a) a heavy chain variable (VH) domain comprising an aminosequence having at least 90% sequence identity to (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or thesequence of, the amino acid sequence of any one of SEQ ID NOs: 36, 47,48, 49, 50, 51, and 52; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 90% identity to (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of any one of SEQID NO: 37, 53, 58, or 59; or (c) a VH domain as in (a) and a VL domainas in (b).

In some instances, any of the preceding antibodies (e.g., anti-tryptaseantibodies) may include one, two, three, or four of the following heavychain framework regions (FRs): (a) an FR-H1 comprising an amino sequencehaving at least 90% sequence identity to (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence of,the amino acid sequence of EVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO:38); (b) an FR-H2 comprising an amino sequence having at least 90%sequence identity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity), or the sequence of, the amino acidsequence of WX₁RQPPGKGLEWIG (SEQ ID NO: 39), wherein X₁ is Ile or Val;(c) an FR-H3 comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofRX₁TISX₂DTSKNQX₃SLKLSSVTAADTAVYX₄CAR (SEQ ID NO: 40), wherein X₁ is Valor Ser, X₂ is Arg or Val, X₃ is Val or Phe, and X₄ is Tyr or Phe; and(d) an FR-H4 comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 41).

For example, in some instances, any of the preceding antibodies (e.g.,anti-tryptase antibodies) may include one, two, three, or four of thefollowing heavy chain FRs: (a) an FR-H1 comprising the amino acidsequence of EVQLVESGPGLVKPSETLSLTCTVSRFSLI (SEQ ID NO: 38); (b) an FR-H2comprising the amino acid sequence of WIRQPPGKGLEWIG (SEQ ID NO: 42);(c) an FR-H3 comprising the amino acid sequence ofRVTISRDTSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO: 43); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 41).

In another example, in some instances, any of the preceding antibodies(e.g., anti-tryptase antibodies) may include one, two, three, or four ofthe following heavy chain FRs: (a) an FR-H1 comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAVSRFSLI (SEQ ID NO: 44); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 45);(c) an FR-H3 comprising the amino acid sequence ofRSTISRDTSKNTVYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 46); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 41).

In another example, in some instances, any of the preceding antibodies(e.g., anti-tryptase antibodies) may include one, two, three, or four ofthe following heavy chain FRs: (a) an FR-H1 comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAVSRFSLI (SEQ ID NO: 44); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 45);(c) an FR-H3 comprising the amino acid sequence ofRSTISRDTSKNTVYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 46); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 41).

In some instances, any of the preceding antibodies (e.g., anti-tryptaseantibodies) may include one, two, three, or four of the following lightchain FRs: (a) an FR-L1 comprising an amino sequence having at least 90%sequence identity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity), or the sequence of, the amino acidsequence of DX₁QX₂TQSPSSLSASVGDRVTITC (SEQ ID NO: 60), wherein X₁ is Ileor Ala, and X₂ is Met or Leu; (b) an FR-L2 comprising an amino sequencehaving at least 90% sequence identity to (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence of,the amino acid sequence of WYQQKPGKX₁PKLLIY (SEQ ID NO: 61), wherein X₁is Ala or Pro; (c) an FR-L3 comprising an amino sequence having at least90% sequence identity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity), or the sequence of, the amino acidsequence of VPSRFSGSGSX₁TDFTLTISSLQPEDFATYX₂C (SEQ ID NO: 62), whereinX₁ is Gly or Glu, and X₂ is Tyr or Phe; and (d) an FR-L4 comprising anamino sequence having at least 90% sequence identity to (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), orthe sequence of, the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 63).

For example, in particular instances, any of the preceding antibodies(e.g., anti-tryptase antibodies) may include one, two, three, or four ofthe following light chain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 64); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 65);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 66); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 63).

In some embodiments, any of the preceding antibodies (e.g.,anti-tryptase antibodies) may include one, two, three, or four of thefollowing light chain FRs: (a) an FR-L1 comprising the amino acidsequence of AAVLTQTPASVSAAVGGTVSISC (SEQ ID NO: 67); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 68);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFKGSGSETQFTLTISDVQX₁DDAATYFC (SEQ ID NO: 69), wherein X₁ is Cys orAla; and (d) an FR-L4 comprising the amino acid sequence of FGQGTKVEIKFGGGTEVVVK (SEQ ID NO: 70).

In some instances, the antibody (e.g., the anti-tryptase antibody)includes (a) a heavy chain variable (VH) domain comprising an aminosequence having at least 90% sequence identity to (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or thesequence of, the amino acid sequence of any one of SEQ ID NOs: 36, 47,48, 49, 50, 51, and 52; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 90% identity to (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of any one of SEQID NO: 37, 53, 58, and 59; or (c) a VH domain as in (a) and a VL domainas in (b). For example, in some instances, the antibody comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 36 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 37. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 47 and a VL domain comprising the amino acidsequence of SEQ ID NO: 37. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 48 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 37. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 49 and a VL domain comprising the amino acidsequence of SEQ ID NO: 37. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 50 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 37. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 50 and a VL domain comprising the amino acidsequence of SEQ ID NO: 37. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 51 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 37. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 52 and a VL domain comprising the amino acidsequence of SEQ ID NO: 37. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 36 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 53. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 47 and a VL domain comprising the amino acidsequence of SEQ ID NO: 53. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 48 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 53. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 49 and a VL domain comprising the amino acidsequence of SEQ ID NO: 53. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 50 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 53. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 51 and a VL domain comprising the amino acidsequence of SEQ ID NO: 53. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 52 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 53. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 36 and a VL domain comprising the amino acidsequence of SEQ ID NO: 58. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 47 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 58. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 48 and a VL domain comprising the amino acidsequence of SEQ ID NO: 58. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 49 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 58. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 50 and a VL domain comprising the amino acidsequence of SEQ ID NO: 58. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 51 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 58. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 36 and a VL domain comprising the amino acidsequence of SEQ ID NO: 59. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 47 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 59. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 48 and a VL domain comprising the amino acidsequence of SEQ ID NO: 59. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 49 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 59. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 50 and a VL domain comprising the amino acidsequence of SEQ ID NO: 59. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 51 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 59.

In another particular example, in some embodiments, the anti-tryptaseantibody may include (a) an HVR-H1 comprising the amino acid sequence ofGYAIT (SEQ ID NO: 30); (b) an HVR-H2 comprising the amino acid sequenceof GISSAATTFYSSWAKS (SEQ ID NO: 31); (c) an HVR-H3 comprising the aminoacid sequence of DPRGYGAALDRLDL (SEQ ID NO: 32); (d) an HVR-L1comprising the amino acid sequence of QSIKSVYNNRLG (SEQ ID NO: 33); (e)an HVR-L2 comprising the amino acid sequence of ETSILTS (SEQ ID NO: 34);and (f) an HVR-L3 comprising the amino acid sequence of AGGFDRSGDTT (SEQID NO: 35). In some embodiments, the anti-tryptase antibody includes (a)a heavy chain variable (VH) domain comprising an amino sequence havingat least 90% sequence identity to (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence of, theamino acid sequence of SEQ ID NO: 36; (b) a light chain variable (VL)domain comprising an amino acid sequence having at least 90% identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of SEQ ID NO: 37;or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the anti-tryptase antibody includes one, two, three, orfour of the following heavy chain framework regions (FRs): (a) an FR-H1comprising the amino acid sequence of EVQLVESGPGLVKPSETLSLTCTVSRFSLI(SEQ ID NO: 38); (b) an FR-H2 comprising the amino acid sequence ofWIRQPPGKGLEWIG (SEQ ID NO: 42); (c) an FR-H3 comprising the amino acidsequence of RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO: 43); and (d) anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 41).In some embodiments, the anti-tryptase antibody includes one, two,three, or four of the following light chain FRs: (a) an FR-L1 comprisingthe amino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 64); (b)an FR-L2 comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ IDNO: 65); (c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 66); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 63). Insome embodiments, the anti-tryptase antibody includes a VH domaincomprising the amino acid sequence of SEQ ID NO: 36 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 37, such as theanti-tryptase antibody huE04.v2.

In another particular example, in some embodiments, the antibody (e.g.,the anti-tryptase antibody) may include (a) an HVR-H1 comprising theamino acid sequence of GYAIT (SEQ ID NO: 30); (b) an HVR-H2 comprisingthe amino acid sequence of GISSAATTFYSSWAKS (SEQ ID NO: 31); (c) anHVR-H3 comprising the amino acid sequence of DPRGYGAALDRLDL (SEQ ID NO:32); (d) an HVR-L1 comprising the amino acid sequence of QSIKSVYNNRLG(SEQ ID NO: 33); (e) an HVR-L2 comprising the amino acid sequence ofETSILTS (SEQ ID NO: 34); and (f) an HVR-L3 comprising the amino acidsequence of AGGFDRSGDTT (SEQ ID NO: 35). In some embodiments, theantibody (e.g., the anti-tryptase antibody) includes (a) a heavy chainvariable (VH) domain comprising an amino sequence having at least 90%sequence identity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity), or the sequence of, the amino acidsequence of SEQ ID NO: 52; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 90% identity to (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of SEQ ID NO: 53;or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody (e.g., the anti-tryptase antibody) includesone, two, three, or four of the following heavy chain framework regions(FRs): (a) an FR-H1 comprising the amino acid sequence ofQXSLEESGGGLFKPTDTLTLTCTVSRFSLI (SEQ ID NO: 54); (b) an FR-H2 comprisingthe amino acid sequence of WVRQSPENGLEWIG (SEQ ID NO: 55); (c) an FR-H3comprising the amino acid sequence of RSTITRNTNENTVTLKMTSLTAADTATYFCAR(SEQ ID NO: 56); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 57). In some embodiments, the antibody (e.g.,the anti-tryptase antibody) includes one, two, three, or four of thefollowing light chain FRs: (a) an FR-L1 comprising the amino acidsequence of AAVLTQTPASVSAAVGGTVSISC (SEQ ID NO: 67); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 68);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFKGSGSETQFTLTISDVQX₁DDAATYFC (SEQ ID NO: 69), wherein X₁ is Cys orAla; and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIKFGGGTEVVVK (SEQ ID NO: 70). In some embodiments, the antibody(e.g., the anti-tryptase antibody) includes a VH domain comprising theamino acid sequence of SEQ ID NO: 52 and a VL domain comprising theamino acid sequence of SEQ ID NO: 53, such as the antibody E104. In someembodiments, the antibody (e.g., the anti-tryptase antibody) includes aVH domain comprising the amino acid sequence of SEQ ID NO: 52 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 59.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofthe amino acid sequence of SEQ ID NO: 76 and/or (b) a light chaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of the amino acidsequence of SEQ ID NO: 77. In some instances, the antibody comprises aheavy chain comprising the amino acids sequence of SEQ ID NO: 76 and alight chain comprising the amino acid sequence of SEQ ID NO: 77. In someembodiments, the heavy chain further comprises a lysine (K) residue atthe C-terminus.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofthe amino acid sequence of SEQ ID NO: 78 and/or (b) a light chaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of the amino acidsequence of SEQ ID NO: 79. In some instances, the antibody comprises aheavy chain comprising the amino acids sequence of SEQ ID NO: 78 and alight chain comprising the amino acid sequence of SEQ ID NO: 79. In someembodiments, the heavy chain further comprises a lysine (K) residue atthe C-terminus.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofthe amino acid sequence of SEQ ID NO: 80 and/or (b) a light chaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of the amino acidsequence of SEQ ID NO: 81. In some instances, the antibody comprises aheavy chain comprising the amino acids sequence of SEQ ID NO: 80 andalight chain comprising the amino acid sequence of SEQ ID NO: 81. Insome embodiments, the heavy chain further comprises a lysine (K) residueat the C-terminus.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising an amino sequence having at least 90% sequenceidentity to (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity), or the sequence of, the amino acid sequence ofthe amino acid sequence of SEQ ID NO: 82 and/or (b) a light chaincomprising an amino sequence having at least 90% sequence identity to(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity), or the sequence of, the amino acid sequence of the amino acidsequence of SEQ ID NO: 83. In some instances, the antibody comprises aheavy chain comprising the amino acids sequence of SEQ ID NO: 82 and alight chain comprising the amino acid sequence of SEQ ID NO: 83. In someembodiments, the heavy chain further comprises a lysine (K) residue atthe C-terminus.

In some instances, any of the preceding antibodies binds to an epitopeon human tryptase beta 1 comprising at least one, at least two, or allthree residues selected from the group consisting of Gln100, Leu101, andLeu102 of SEQ ID NO: 71. In some embodiments, the epitope on humantryptase beta 1 further comprises one or more amino acid residuesselected from the group consisting of Trp55, Gln67, Asp82, Leu83, Ala84,Arg87, Pro103, Val104, Ser105, Arg106, Glu126, Leu127, Glu128, andGlu129 of SEQ ID NO: 71. In some embodiments, the epitope on humantryptase beta 1 comprises at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,or all fourteen amino acid residues selected from the group consistingof Trp55, Gln67, Asp82, Leu83, Ala84, Arg87, Pro103, Val104, Ser105,Arg106, Glu126, Leu127, Glu128, and Glu129 of SEQ ID NO: 71. In someembodiments, the epitope comprises Gln35, Trp55, Gln67, Asp82, Leu83,Ala84, Arg87, Gln100, Leu101, Leu102, Pro103, Val104, Ser105, Arg106,Glu126, Leu127, Glu128, Glu129, and Arg216 of SEQ ID NO: 71. In someembodiments, the epitope is relative to a human tryptase beta 1 monomeror tetramer. In some embodiments, the epitope is relative to a humantryptase beta 1 tetramer, and the epitope on human tryptase beta 1further comprises one or both of Gln35 and Arg216 of SEQ ID NO: 71. Insome embodiments, the epitope is determined by an X-ray crystallographymodel. In some embodiments, the antibody is capable of dissociating thesmall interface and/or the large interface of human tryptase beta 1.

In some instances, any of the preceding anti-tryptase antibodiesincludes a paratope that binds tryptase (e.g., human tryptase beta 1)that includes one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 19 amino acid residues) selectedfrom the group consisting of light chain variable region amino acidresidues Tyr29; Asn30; Arg32; and Arg94 and the heavy chain variableregion amino acid residues Gly31; Tyr32; Ser52; Ser53; Ala54; Thr56;Phe58; Pro96; Arg97; Gly98; Tyr99; and Arg100e.

For example, in some instances, the anti-tryptase antibody includes aparatope that binds tryptase (e.g., human tryptase beta 1) that includeslight chain variable region amino acid residues Tyr29; Asn30; Arg32; andArg94 or the heavy chain variable region amino acid residues Gly31;Tyr32; Ser52; Ser53; Ala54; Thr56; Phe58; Pro96; Arg97; Gly98; Tyr99;and Arg100e. In some instances, the anti-tryptase antibody includes aparatope that binds tryptase (e.g., human tryptase beta 1) that includeslight chain variable region amino acid residues Tyr29; Asn30; Arg32; andArg94 and the heavy chain variable region amino acid residues Gly31;Tyr32; Ser52; Ser53; Ala54; Thr56; Phe58; Pro96; Arg97; Gly98; Tyr99;and Arg100e.

In some instances, any of the preceding anti-tryptase antibodies bindshuman tryptase. In some instances, any of the preceding antibodies bindscynomolgus monkey (cyno) tryptase. In some instances, the antibody bindshuman tryptase alpha or human tryptase beta. In some instances, theantibody binds human tryptase beta 1, human tryptase beta 2, or humantryptase beta 3.

In another aspect, the invention provides anti-tryptase antibodies thatbind to an epitope on tryptase (e.g., human tryptase beta 1) thatincludes one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, or 7amino acid residues) selected from the group consisting of His51, Val80,Lys81, Asp82, Leu83, Ala84, and Ala85, which may be in reference to theamino acid sequence of SEQ ID NO: 71 or a corresponding amino acid ofany tryptase protein. For example, in some embodiments, the antibodybinds to an epitope on tryptase (e.g., human tryptase beta 1) comprisingat least one, at least two, at least three, or all four residuesselected from the group consisting of His51, Val80, Lys81, and Asp82 ofSEQ ID NO: 71 or a corresponding amino acid of any tryptase protein. Insome embodiments, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) comprising His51 and at least one, at least two,or all three residues selected from the group consisting of Val 80,Lys81, and Asp82 of SEQ ID NO: 71 or a corresponding amino acid of anytryptase protein. In some embodiments, the epitope on tryptase (e.g.,human tryptase beta 1) further comprises one or more amino acid residuesselected from the group consisting of Gln67, Leu83, Ala84, Ala85, Arg87,Pro103, Val104, Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71 or a corresponding amino acid of any tryptase protein. In someembodiments, the epitope on tryptase (e.g., human tryptase beta 1)comprises at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, or all twelve amino acid residues selected from thegroup consisting of Gln67, Leu83, Ala84, Ala85, Arg87, Pro103, Val104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some embodiments,the epitope on tryptase (e.g., human tryptase beta 1) comprises His51,Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Va104,Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO:71 or acorresponding amino acid of any tryptase protein. In some embodiments,the epitope is relative to a tryptase (e.g., human tryptase beta 1)monomer or tetramer. In some embodiments, the epitope is determined byan X-ray crystallography model. In some embodiments, the antibody iscapable of dissociating both the small interface of tetrameric tryptaseand the large interface of tetrameric tryptase tryptase (e.g., humantryptase beta 1).

In yet another aspect, the invention provides anti-tryptase antibodiesthat binds to an epitope on tryptase (e.g., human tryptase beta 1) thatincludes one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, or 7amino acid residues) elected from the group consisting of Gln100,Leu101, Leu102, Pro103, Val104, Ser105, and Arg106, which may be inreference to the amino acid sequence of SEQ ID NO: 71 or a correspondingamino acid of any tryptase protein. In some embodiments, the epitope ontryptase (e.g., human tryptase beta 1) further comprises one or moreamino acid residues selected from the group consisting of Trp55, Gln67,Asp82, Leu83, Ala84, Arg87, Pro103, Va104, Ser105, Arg106, Glu126,Leu127, Glu128, and Glu129 of SEQ ID NO: 71 or a corresponding aminoacid of any tryptase protein. In some embodiments, the epitope ontryptase (e.g., human tryptase beta 1) comprises at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, or all fourteen amino acid residues selectedfrom the group consisting of Trp55, Gln67, Asp82, Leu83, Ala84, Arg87,Pro103, Val104, Ser105, Arg106, Glu126, Leu127, Glu128, and Glu129 ofSEQ ID NO: 71 or a corresponding amino acid of any tryptase protein. Insome embodiments, the epitope comprises Gln35, Trp55, Gln67, Asp82,Leu83, Ala84, Arg87, Gln100, Leu101, Leu102, Pro103, Val104, Ser105,Arg106, Glu126, Leu127, Glu128, Glu129, and Arg216 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some embodiments,the epitope is relative to a tryptase (e.g., human tryptase beta 1)monomer or tetramer. In some embodiments, the epitope is relative to atetramer, and the epitope on tryptase (e.g., human tryptase beta 1)further comprises one or both of Gln35 and Arg216 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some embodiments,the epitope is determined by an X-ray crystallography model. In someembodiments, the antibody is capable of dissociating the small interfaceand/or the large interface of tryptase (e.g., human tryptase beta 1).

In some embodiments, any of the preceding antibodies binds to an epitopeon tryptase (e.g., human tryptase beta 1) that includes one or moreamino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 aminoacid residues) selected from the group consisting of Gln67, Asp82,Leu83, Ala84, Arg87, Pro103, Val104, Ser105, Arg106, Glu128, and Glu129,which may be in reference to the amino acid sequence of SEQ ID NO: 71 ora corresponding amino acid of any tryptase protein.

For example, in some instances, any of the preceding antibodies binds toan epitope on tryptase (e.g., human tryptase beta 1) that includes Gln67of SEQ ID NO: 71 or a corresponding amino acid of any tryptase protein.In some instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that includes Asp82 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some instances, theantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that includes Leu83 of SEQ ID NO: 71 or a corresponding amino acid ofany tryptase protein. In some instances, the antibody binds to anepitope on tryptase (e.g., human tryptase beta 1) that includes Ala84 ofSEQ ID NO: 71 or a corresponding amino acid of any tryptase protein. Insome instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that includes Arg87 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some instances, theantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that includes Pro103 of SEQ ID NO: 71 or a corresponding amino acid ofany tryptase protein. In some instances, the antibody binds to anepitope on tryptase (e.g., human tryptase beta 1) comprising Val104 ofSEQ ID NO: 71 or a corresponding amino acid of any tryptase protein. Insome instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that includes Ser105 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some instances, theantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that includes Arg106 of SEQ ID NO: 71 or a corresponding amino acid ofany tryptase protein. In some instances, the antibody binds to anepitope on tryptase (e.g., human tryptase beta 1) that includes Glu128of SEQ ID NO: 71 or a corresponding amino acid of any tryptase protein.In some instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that includes Glu129 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein.

In some embodiments, any of the preceding antibodies binds to an epitopeon tryptase (e.g., human tryptase beta 1) that includes two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, or all eleven amino acidresidues selected from the group consisting of Gln67, Asp82, Leu83,Ala84, Arg87, Pro103, Val104, Ser105, Arg106, Glu128, and Glu129, whichmay be in reference to the amino acid sequence of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein.

In some instances, any of the preceding antibodies binds to an epitopeon tryptase (e.g., human tryptase beta 1) that further includes one ormore additional amino acid residues (e.g., 1, 2, 3, 4, or 5 additionalamino acid residues) selected from the group consisting of His51, Val80,Lys81, Ala85, and Pro130, which may be in reference to the amino acidsequence of SEQ ID NO: 71 or a corresponding amino acid of any tryptaseprotein. For example, in some instances, the antibody binds to anepitope on tryptase (e.g., human tryptase beta 1) that further includesHis51 of SEQ ID NO: 71 or a corresponding amino acid of any tryptaseprotein. In some instances, the antibody binds to an epitope on tryptase(e.g., human tryptase beta 1) that further includes Val80 of SEQ ID NO:71 or a corresponding amino acid of any tryptase protein. In someembodiments the anti-tryptase antibody binds to an epitope on tryptase(e.g., human tryptase beta 1) that further includes Lys81 of SEQ ID NO:71 or a corresponding amino acid of any tryptase protein. In someinstances, the antibody binds to an epitope on tryptase (e.g., humantryptase beta 1) that further includes Ala85 of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein. In some instances, theantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that further includes Pro130 of SEQ ID NO: 71 or a corresponding aminoacid of any tryptase protein.

In some instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that further includes two or more, three or more,four or more, or five or more additional amino acid residues selectedfrom the group consisting of His51, Val80, Lys81, Ala85, and Pro130,which may be in reference to the amino acid sequence of SEQ ID NO: 71 ora corresponding amino acid of any tryptase protein.

In some instances, the anti-tryptase antibody binds to an epitope ontryptase (e.g., human tryptase beta 1) that includes His51, Gln67,Val80, Lys81, Asp82, Leu83, Ala84, Ala85, Arg87, Pro103, Val104, Ser105,Arg106, Glu128, Glu129, and Pro130 of SEQ ID NO: 71, which may be inreference to the amino acid sequence of SEQ ID NO: 71 or a correspondingamino acid of any tryptase protein. In some instances, the anti-tryptaseantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that consists of His51, Gln67, Val80, Lys81, Asp82, Leu83, Ala84, Ala85,Arg87, Pro103, Val104, Ser105, Arg106, Glu128, Glu129, and Pro130 of SEQID NO: 71.

In other instances, any of the preceding anti-tryptase antibodies bindsto an epitope on tryptase (e.g., human tryptase beta 1) that furtherincludes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) additional aminoacid residues selected from the group consisting of Gln35, Trp55,Gln100, Leu101, Leu102, Glu126, Leu127, and Arg216, which may be inreference to the amino acid sequence of SEQ ID NO: 71 or a correspondingamino acid of any tryptase protein. For example, in some instances, theantibody binds to an epitope on tryptase (e.g., human tryptase beta 1)that further includes Gln35 of SEQ ID NO: 71 or a corresponding aminoacid of any tryptase protein. In some instances, the antibody binds toan epitope on tryptase (e.g., human tryptase beta 1) that furtherincludes Trp55 of SEQ ID NO: 71 or a corresponding amino acid of anytryptase protein. In some instances, the antibody binds to an epitope ontryptase (e.g., human tryptase beta 1) that further includes Gln100 ofSEQ ID NO: 71 or a corresponding amino acid of any tryptase protein. Insome instances, the antibody binds to an epitope on tryptase (e.g.,human tryptase beta 1) that further includes Leu101 of SEQ ID NO: 71 ora corresponding amino acid of any tryptase protein. In some instances,the antibody binds to an epitope on tryptase (e.g., human tryptasebeta 1) that further includes Leu102 of SEQ ID NO: 71 or a correspondingamino acid of any tryptase protein. In some instances, the antibodybinds to an epitope on tryptase (e.g., human tryptase beta 1) thatfurther includes Glu126 of SEQ ID NO: 71 or a corresponding amino acidof any tryptase protein. In some instances, the antibody binds to anepitope on tryptase (e.g., human tryptase beta 1) that further includesLeu127 of SEQ ID NO: 71 or a corresponding amino acid of any tryptaseprotein. In some instances, the antibody binds to an epitope on tryptase(e.g., human tryptase beta 1) that further includes Arg216 of SEQ ID NO:71 or a corresponding amino acid of any tryptase protein. In someinstances, the antibody binds to an epitope on tryptase (e.g., humantryptase beta 1) that further includes two or more, three or more, fouror more, five or more, six or more, seven or more, or eight or moreadditional amino acid residues selected from the group consisting ofGln35, Trp55, Gln100, Leu101, Leu102, Glu126, Leu127, and Arg216, whichmay be in reference to the amino acid sequence of SEQ ID NO: 71 or acorresponding amino acid of any tryptase protein.

In some instances, the anti-tryptase antibody binds to an epitope ontryptase (e.g., human tryptase beta 1) that includes Gln35, Trp55,Gln67, Asp82, Leu83, Ala84, Arg87, Gln100, Leu101, Leu102, Pro103,Val104, Ser105, Arg106, Glu126, Leu127, Glu128, Glu129, and Arg216,which may be in reference to the amino acid sequence of SEQ ID NO: 71 ora corresponding amino acid of any tryptase protein. In some instances,the anti-tryptase antibody binds to an epitope on tryptase (e.g., humantryptase beta 1) that consists of Gln35, Trp55, Gln67, Asp82, Leu83,Ala84, Arg87, Gln100, Leu101, Leu102, Pro103, Val104, Ser105, Arg106,Glu126, Leu127, Glu128, Glu129, and Arg216 of SEQ ID NO: 71.

In another aspect, the invention provides an antibody that competes forbinding to tryptase (e.g., human tryptase beta 1) with any of thepreceding antibodies.

In another aspect, the invention provides an antibody that binds to thesame epitope or an overlapping epitope as any of the precedingantibodies.

In some embodiments, any of the preceding antibodies may have theability to disrupt tryptase having a tetrameric structure (such asmature tryptase present in, or released from, mast cell secretorygranules) to form smaller molecular weight species, e.g., monomers,dimers, and/or trimers.

In a further aspect, an antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, ≤1pM, or ≤0.1 pM (e.g., 10⁻⁶ M or less, e.g., from 10⁻⁶ M to 10⁻⁹ M orless, e.g., from 10⁻⁹ M to 10⁻¹³ M or less). In some embodiments, ananti-tryptase antibody of the invention binds to tryptase (e.g., humantryptase, e.g., human tryptase beta) with a K_(D) of about 100 nM orlower (e.g., 100 nM or lower, 10 nM or lower, 1 nM or lower, 100 pM orlower, 10 pM or lower, 1 pM or lower, or 0.1 pM or lower). In someembodiments, the antibody binds tryptase (e.g., human tryptase, e.g.,human tryptase beta) with a K_(D) of 10 nM or lower (e.g., 10 nM orlower, 1 nm or lower, 100 pM or lower, 10 pM or lower, 1 pM or lower, or0.1 pM or lower). In some embodiments, the antibody binds tryptase(e.g., human tryptase, e.g., human tryptase beta) with a K_(D) of 1 nMor lower (e.g., 1 nm or lower, 100 pM or lower, 10 pM or lower, 1 pM orlower, or 0.1 pM or lower). In some embodiments, any of theanti-tryptase antibodies described above or herein binds to tryptase(e.g., human tryptase, e.g., human tryptase beta) with a K_(D) of about0.5 nM or lower (e.g., 0.5 nm or lower, 400 pM or lower, 300 pM orlower, 200 pM or lower, 100 pM or lower, 50 pM or lower, 25 pM or lower,10 pM or lower, 1 pM or lower, or 0.1 pM or lower). In some embodiments,the antibody binds tryptase (e.g., human tryptase, e.g., human tryptasebeta) with a K_(D) between about 0.1 nM to about 0.5 nM (e.g., about 0.1nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, or about 0.5 nM). In someembodiments, the antibody binds tryptase (e.g., human tryptase, e.g.,human tryptase beta) with a K_(D) of about 0.4 nM. In some embodiments,the antibody binds tryptase (e.g., human tryptase, e.g., human tryptasebeta) with a K_(D) of about 0.18 nM.

In one embodiment, K_(D) is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al. J. Mol.Biol. 293:865-881, 1999). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al. Cancer Res. 57:4593-4599, 1997). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN®-20) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, K_(D) is measured using a BIACORE®surface plasmon resonance assay. For example, an assay using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) isperformed at 25° C. with immobilized antigen CM5 chips at ˜10 responseunits (RU). In one embodiment, carboxymethylated dextran biosensor chips(CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2pM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in phosphate buffered saline (PBS) with0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al. (J. Mol. Biol.293:865-881, 1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

In some embodiments, K_(D) is measured using a BIACORE® SPR assay, forexample, as described in Example 1, Section (A)(vii). In someembodiments, the SPR assay can use a BIAcore® T200 or an equivalentdevice. In some embodiments, BIAcore® Series S CM5 sensor chips (orequivalent sensor chips) are immobilized with monoclonal mouseanti-human IgG (Fc) antibody and anti-tryptase antibodies aresubsequently captured on the flow cell. Serial 3-fold dilutions of theHis-tagged human tryptase beta 1 monomer (SEQ ID NO: 128) are injectedat a flow rate of 30 μl/min. Each sample is analyzed with 3 minassociation and 10 min dissociation. The assay is performed at 25° C.After each injection, the chip is regenerated using 3 M MgCl₂. Bindingresponse is corrected by subtracting the response units (RU) from a flowcell capturing an irrelevant IgG at similar density. A 1:1 Languir modelof simultaneous fitting of k_(on) and k_(off) is used for kineticsanalysis.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9:129-134, 2003; and Hollinger et al. Proc.Natl. Acad. Sci. USA 90: 6444-6448, 1993. Triabodies and tetrabodies arealso described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (see, e.g.,U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA,81:6851-6855, 1984). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs (or portions thereof) are derivedfrom a non-human antibody, and FRs (or portions thereof) are derivedfrom human antibody sequences. A humanized antibody optionally will alsocomprise at least a portion of a human constant region. In someembodiments, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (e.g., theantibody from which the HVR residues are derived), for example, torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro et al. Front. Biosci. 13:1619-1633, 2008, and arefurther described, e.g., in Riechmann et al. Nature 332:323-329, 1988;Queen et al. Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989; U.S. Pat.Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al.Methods 36:25-34, 2005 (describing specificity determining region (SDR)grafting); Padlan, Mol. Immunol. 28:489-498, 1991 (describing“resurfacing”); Dall'Acqua et al. Methods 36:43-60, 2005 (describing “FRshuffling”); and Osbourn et al. Methods 36:61-68, 2005 and Klimka et al.Br. J. Cancer, 83:252-260, 2000 (describing the “guided selection”approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285, 1992; and Presta etal. J. Immunol., 151:2623, 1993); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro et al. Front. Biosci. 13:1619-1633, 2008); and framework regionsderived from screening FR libraries (see, e.g., Baca et al. J. Biol.Chem. 272:10678-10684, 1997 and Rosok et al. J. Biol. Chem.271:22611-22618, 1996).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk et al. Curr.Opin. Pharmacol. 5:368-74, 2001 and Lonberg, Curr. Opin. Immunol.20:450-459, 2008.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125, 2005. Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology. Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol. 133:3001, 1984; Brodeur et al. Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987); and Boerner et al. J. Immunol. 147: 86, 1991). Human antibodiesgenerated via human B-cell hybridoma technology are also described in Liet al. Proc. Natl. Acad. Sci. USA, 103:3557-3562, 2006. Additionalmethods include those described, for example, in U.S. Pat. No. 7,189,826(describing production of monoclonal human IgM antibodies from hybridomacell lines) and Ni, Xiandai Mianyixue, 26(4):265-268, 2006 (describinghuman-human hybridomas). Human hybridoma technology (Trioma technology)is also described in Vollmers et al. Histology and Histopathology20(3):927-937, 2005 and Vollmers et al. Methods and Findings inExperimental and Clinical Pharmacology 27(3):185-91, 2005.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al. Nature 348:552-554, 1990; Clackson et al.Nature 352: 624-628, 1991; Marks et al. J. Mol. Biol. 222: 581-597,1992; Marks et al. in Methods in Molecular Biology 248:161-175 (Lo, ed.,Human Press, Totowa, N.J., 2003); Sidhu et al. J. Mol. Biol. 338(2):299-310, 2004; Lee et al. J. Mol. Biol. 340(5): 1073-1093, 2004;Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472, 2004; and Leeet al. J. Immunol. Methods 284(1-2): 119-132, 2004.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. Ann. Rev. Immunol.,12: 433-455, 1994. Phage typically display antibody fragments, either assingle-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaïve repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al. EMBO J. 12:725-734, 1993. Finally, naïve libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable HVR3regions and to accomplish rearrangement in vitro, as described byHoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publicationsdescribing human antibody phage libraries include, for example: U.S.Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574,2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764,2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In certain embodiments, bispecific antibodies maybind to two different epitopes of tryptase. In certain embodiments, oneof the binding specificities is for tryptase and the other is for anyother antigen (e.g., a second biological molecule). In some embodiments,bispecific antibodies may bind to two different epitopes of tryptase. Inother embodiments, one of the binding specificities is for tryptase(e.g., human tryptase, e.g., human tryptase beta) and the other is forany other antigen (e.g., a second biological molecule, e.g., IL-13,IL-4, IL-5, IL-17, IL-33, IgE, M1 prime, CRTH2, or TRPA). Accordingly,the bispecific antibody may have binding specificity for tryptase andIL-13; tryptase and IL-4; tryptase and IL-5; tryptase and IL-17, ortryptase ant IL-33. In particular, the bispecific antibody may havebinding specificity for tryptase and IL-13 or tryptase and IL-33.Bispecific antibodies can be prepared as full length antibodies orantibody fragments.

For example, in some instances, a bispecific antibody includes a firstbinding domain that binds tryptase and a second binding domain thatbinds IL-13. In some embodiments, the first binding domain that bindstryptase may include, for example, at least one, two, three, four, five,or six hypervariable regions (HVRs) selected from: (a) an HVR-H1comprising the amino acid sequence of X₁X₂GMX₃ (SEQ ID NO: 1), whereinX₁ is Asp or Ser, X₂ is Tyr or Phe, and X₃ is Val or His; (b) an HVR-H2comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2);(c) an HVR-H3 comprising the amino acid sequence of RX₁X₂X₃DWYFDV (SEQID NO: 3), wherein X₁ is Asn or Asp, X₂ is Tyr or Asn, and X₃ is Asp orTyr; (d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQID NO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS(SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6), or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 1-6. In some instances, the second binding domain that binds toIL-13 may, for example, include at least one, two, three, four, five, orsix HVRs selected from (a) HVR-H1 comprising the amino acid sequence ofAYSVN (SEQ ID NO: 84); (b) HVR-H2 comprising the amino acid sequence ofMIWGDGKIVYNSALKS (SEQ ID NO: 85); (c) HVR-H3 comprising the amino acidsequence of DGYYPYAMDN (SEQ ID NO: 86); (d) HVR-L1 comprising the aminoacid sequence of RASKSVDSYGNSFMH (SEQ ID NO: 87); (e) HVR-L2 comprisingthe amino acid sequence of LASNLES (SEQ ID NO: 88); and (f) HVR-L3comprising the amino acid sequence of QQNNEDPRT (SEQ ID NO: 89), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 84-89. In someembodiments, the second binding domain comprises one, two, three, four,five, or six HVRs of the anti-IL-13 antibody lebrikizumab.

For example, in some instances, the first binding domain that bindstryptase comprises at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from: (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6), such as hu31a.v11. In some instances, thesecond binding domain that binds IL-13 may, for example, comprise atleast one, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of AYSVN (SEQ ID NO: 84); (b) HVR-H2comprising the amino acid sequence of MIWGDGKIVYNSALKS (SEQ ID NO: 85);(c) HVR-H3 comprising the amino acid sequence of DGYYPYAMDN (SEQ ID NO:86); (d) HVR-L1 comprising the amino acid sequence of RASKSVDSYGNSFMH(SEQ ID NO: 87); (e) HVR-L2 comprising the amino acid sequence ofLASNLES (SEQ ID NO: 88); and (f) HVR-L3 comprising the amino acidsequence of QQNNEDPRT (SEQ ID NO: 89). In some embodiments, the secondbinding domain comprises one, two, three, four, five, or six HVRs of theanti-IL-13 antibody lebrikizumab. In some embodiments, the first bindingdomain comprises the amino acid sequence of the VH and/or VL ofhu31a.v11 and the second binding domain comprises the amino acidsequence of the VH and/or VL of the anti-IL-13 antibody lebrikizumab.

Any of the preceding bispecific anti-tryptase/anti-IL-13 antibodies mayinclude a first binding domain that binds tryptase comprising (a) a VHdomain comprising an amino acid sequence having at least 80% sequenceidentity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, orthe sequence of, SEQ ID NO: 9; (b) a VL domain comprising an amino acidsequence having at least 80% sequence identity (e.g., 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:10; or (c) a VH domain as in (a) and a V_(L) domain as in (b). Any ofthe preceding bispecific anti-tryptase/anti-IL-13 antibodies may includea second binding domain that binds to IL-13 comprising (a) a VH domaincomprising an amino acid sequence having at least 80% sequence identity(e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 90 or SEQ ID NO: 114; (b) a VL domain comprisingan amino acid sequence having at least 80% sequence identity (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,SEQ ID NO: 91 or SEQ ID NO: 115; or (c) a VH domain as in (a) and a VLdomain as in (b). In some instances, the second binding domain comprisesthe VH and/or VL domain of lebrikizumab.

In another example, in some instances, a bispecific antibody includes afirst binding domain that binds tryptase and a second binding domainthat binds IL-33. The second binding domain that binds IL-33 may includeany of the anti-IL-33 antibodies described, for example, in U.S. PatentPublication No. 2016/0168242, which is incorporated herein by referencein its entirety. In some embodiments, the first binding domain thatbinds tryptase may include, for example, at least one, two, three, four,five, or six hypervariable regions (HVRs) selected from: (a) an HVR-H1comprising the amino acid sequence of X₁X₂GMX₃ (SEQ ID NO: 1), whereinX₁ is Asp or Ser, X₂ is Tyr or Phe, and X₃ is Val or His; (b) an HVR-H2comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2);(c) an HVR-H3 comprising the amino acid sequence of RX₁X₂X₃DWYFDV (SEQID NO: 3), wherein X₁ is Asn or Asp, X₂ is Tyr or Asn, and X₃ is Asp orTyr; (d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQID NO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS(SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6), or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 1-6. In some instances, the second binding domain that binds toIL-33 may, for example, include at least one, two, three, four, five, orsix HVRs selected from (a) HVR-H1 comprising the amino acid sequence ofSFSMS (SEQ ID NO: 120); (b) HVR-H2 comprising the amino acid sequence ofTISGGKTFTDYVDSVKG (SEQ ID NO: 121); (c) HVR-H3 comprising the amino acidsequence of ANYGNWFFEV (SEQ ID NO: 122); (d) HVR-L1 comprising the aminoacid sequence of RASESVAKYGLSLLN (SEQ ID NO: 123); (e) HVR-L2 comprisingthe amino acid sequence of AASNRGS (SEQ ID NO: 124); and (f) HVR-L3comprising the amino acid sequence of QQSKEVPFT (SEQ ID NO: 125), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 120-125. In someembodiments, the second binding domain comprises one, two, three, four,five, or six HVRs of the anti-IL-33 antibody 10C12.38.H6.87Y.58I.

For example, in some instances, the first binding domain that bindstryptase comprises at least one, two, three, four, five, or sixhypervariable regions (HVRs) selected from: (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6), such as hu31a.v11. In some instances, thesecond binding domain that binds IL-33 may, for example, comprise atleast one, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SFSMS (SEQ ID NO: 120); (b) HVR-H2comprising the amino acid sequence of TISGGKTFTDYVDSVKG (SEQ ID NO:121); (c) HVR-H3 comprising the amino acid sequence of ANYGNWFFEV (SEQID NO: 122); (d) HVR-L1 comprising the amino acid sequence ofRASESVAKYGLSLLN (SEQ ID NO: 123); (e) HVR-L2 comprising the amino acidsequence of AASNRGS (SEQ ID NO: 124); and (f) HVR-L3 comprising theamino acid sequence of QQSKEVPFT (SEQ ID NO: 125). In some embodiments,the second binding domain comprises one, two, three, four, five, or sixHVRs of the anti-IL-33 antibody 10C12.38.H6.87Y.581. In someembodiments, the first binding domain comprises the amino acid sequenceof the VH and/or VL of hu31a.v11 and the second binding domain comprisesthe amino acid sequence of the VH and/or VL of the anti-IL-33 antibody10C12.38.H6.87Y.58I.

Any of the preceding bispecific anti-tryptase/anti-IL-33 antibodies mayinclude a first binding domain that binds tryptase comprising (a) a VHdomain comprising an amino acid sequence having at least 80% sequenceidentity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, orthe sequence of, SEQ ID NO: 9; (b) a VL domain comprising an amino acidsequence having at least 80% sequence identity (e.g., 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:10; or (c) a VH domain as in (a) and a VL domain as in (b). Any of thepreceding bispecific anti-tryptase/anti-IL-33 antibodies may include asecond binding domain that binds to IL-33 comprising (a) a VH domaincomprising an amino acid sequence having at least 80% sequence identity(e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 126; (b) a VL domain comprising an amino acidsequence having at least 80% sequence identity (e.g., 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:127; or (c) a VH domain as in (a) and a VL domain as in (b). In someinstances, the second binding domain comprises the VH and/or VL domainof 10C12.38.H6.87Y.58I.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein etal. Nature 305: 537, 1983; WO 93/08829; and Traunecker et al. EMBO J.10: 3655, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Pat.No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al. Science, 229: 81, 1985); using leucine zippers to producebispecific antibodies (see, e.g., Kostelny et al. J. Immunol.,148(5):1547-1553, 1992); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al. Proc. Natl.Acad. Sci. USA 90:6444-6448, 1993); and using single-chain Fv (scFv)dimers (see, e.g., Gruber et al. J. Immunol. 152:5368, 1994); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60, 1991.

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to tryptase as wellas another, different antigen (see, US 2008/0069820, for example).

Knobs-into-Holes

The use of knobs-into-holes as a method of producing multispecificantibodies is described, e.g., in U.S. Pat. No. 5,731,168,WO2009/089004, US2009/0182127, US2011/0287009, Marvin and Zhu, ActaPharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) ActaPharmacol. Sin. 26:1-9. A brief nonlimiting discussion is providedbelow.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.,the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g., by alteringnucleic acid encoding the interface). In some embodiments, a nucleicacid encoding the interface of the first polypeptide is altered toencode the protuberance. To achieve this, the nucleic acid encoding atleast one “original” amino acid residue in the interface of the firstpolypeptide is replaced with nucleic acid encoding at least one “import”amino acid residue which has a larger side chain volume than theoriginal amino acid residue. It will be appreciated that there can bemore than one original and corresponding import residue. The side chainvolumes of the various amino residues are shown, for example, in Table 1of US 2011/0287009 or Table 1 of U.S. Pat. No. 7,642,228.

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiments, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine, or valine. See, for example, U.S. Pat. No. 7,642,228.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g., by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T), and valine (V). In some embodiments, animport residue is serine, alanine, or threonine. In some embodiments,the original residue for the formation of the cavity has a large sidechain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe, and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely-accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region isT366W. In some embodiments, a hole mutation in an IgG1 constant regioncomprises one or more mutations selected from T366S, L368A, and Y407V.In some embodiments, a hole mutation in an IgG1 constant regioncomprises T366S, L368A, and Y407V.

In some embodiments, a knob mutation in an IgG4 constant region isT366W. In some embodiments, a hole mutation in an IgG4 constant regioncomprises one or more mutations selected from T366S, L368A, and Y407V.In some embodiments, a hole mutation in an IgG4 constant regioncomprises T366S, L368A, and Y407V.

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody, such as inhibitory activity. Amino acid sequence variants ofan antibody may be prepared by introducing appropriate modificationsinto the nucleotide sequence encoding the antibody, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of residues within the aminoacid sequences of the antibody. Any combination of deletion, insertion,and substitution can be made to arrive at the final construct, providedthat the final construct possesses the desired characteristics, forexample, antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and EFRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened foradesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, for example, using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196, 2008), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al. ed., Human Press, Totowa,N.J., 2001). In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. HVR-H3 and HVR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham et al. Science 244:1081-1085,1989. In this method, a residue or group of target residues (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) are identified andreplaced by a neutral or negatively charged amino acid (e.g., Ala orpolyalanine) to determine whether the interaction of the antibody withantigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, for example, Wright et al. TIBTECH 15:26-32, 1997. Theoligosaccharide may include various carbohydrates, for example, mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. 2003/0157108 and 2004/0093621.Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol.Biol. 336:1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004. Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986; US2003/0157108; and WO 2004/056312 A1, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004; Kanda et al. Biotechnol. Bioeng. 94(4):680-688, 2006; and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch et al. Annu. Rev. Immunol. 9:457-492, 1991.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.,Hellstrom et al. Proc. Natl. Acad. Sci. USA 83:7059-7063, 1986 andHellstrom et al. Proc. Natl. Acad. Sci. USA 82:1499-1502, 1985; U.S.Pat. No. 5,821,337 (see Bruggemann et al. J. Exp. Med. 166:1351-1361,1987). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. Proc. Natl. Acad. Sci. USA 95:652-656, 1998. C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, e.g., Gazzano-Santoro etal. J. Immunol. Methods 202:163, 1996; Cragg et al. Blood 101:1045-1052,2003; and Cragg et al. Blood 103:2738-2743, 2004). FcRn binding and invivo clearance/half life determinations can also be performed usingmethods known in the art (see, e.g., Petkova et al. Intl. Immunol.18(12):1759-1769,2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; andShields et al. J. Biol. Chem. 9(2): 6591-6604, 2001).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), for example, as described inU.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol.164: 4178-4184, 2000.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al. J. Immunol. 117:587, 1976 andKim et al. J. Immunol. 24:249, 1994), are described in US2005/0014934.Those antibodies comprise an Fc region with one or more substitutionstherein which improve binding of the Fc region to FcRn. Such Fc variantsinclude those with substitutions at one or more of Fc region residues:238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fcregion residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan et al. Nature 322:738-40, 1988; U.S. Pat. Nos. 5,648,260and 5,624,821; and WO 94/29351 concerning other examples of Fc regionvariants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, for example, “thioMAbs,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and S400 (EU numbering) of the heavy chain Fcregion. Cysteine engineered antibodies may be generated as described,e.g., in U.S. Pat. No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,and the like.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al. Proc. Natl. Acad. Sci. USA 102: 11600-11605, 2005).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,for example, as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-tryptase antibody describedherein is provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the V_(L) of the antibody and an amino acid sequencecomprising the VH of the antibody, or (2) a first vector comprising anucleic acid that encodes an amino acid sequence comprising the VL ofthe antibody and a second vector comprising a nucleic acid that encodesan amino acid sequence comprising the VH of the antibody. In oneembodiment, the host cell is eukaryotic, for example, a Chinese HamsterOvary (CHO) cell, 293 cell, or lymphoid cell (e.g., Y0, NS0, Sp20 cell).In one embodiment, a method of making an anti-tryptase antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-tryptase antibody, nucleic acidencoding an antibody, for example, as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, for example, U.S.Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli. After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross Nat. Biotech. 22:1409-1414, 2004 and Li et al.Nat. Biotech. 24:210-215, 2006.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, for example,U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al. J. Gen Virol. 36:59, 1977);baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather Biol. Reprod. 23:243-251, 1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al. Proc.Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0,NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki et al. Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268, 2003.

C. Assays

Anti-tryptase antibodies provided herein may be identified, screenedfor, or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an anti-tryptase antibody of the invention is tested forits antigen-binding activity, for example, by known methods such asELISA, Western blot, surface plasmon resonance (SPR), and the like.

In another aspect, competition assays may be used to identify anantibody that competes with an anti-tryptase antibody of the inventionfor binding to tryptase. In certain embodiments, such a competingantibody binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by an anti-tryptase antibody of the invention. Incertain embodiments, such a competing antibody binds to an overlappingepitope (e.g., a linear or a conformational epitope) that is bound by ananti-tryptase antibody of the invention. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris“Epitope Mapping Protocols,” in Methods in Molecular Biology Vol. 66(Humana Press, Totowa, N.J.), 1996.

In an exemplary competition assay, immobilized tryptase is incubated ina solution comprising a first labeled antibody that binds to tryptaseand a second unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to tryptase. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized tryptase is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody totryptase, excess unbound antibody is removed, and the amount of labelassociated with immobilized tryptase is measured. If the amount of labelassociated with immobilized tryptase is substantially reduced in thetest sample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding totryptase. See Harlow et al. Antibodies: A Laboratory Manual Ch.14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.), 1988.

In another embodiment, the competition of two antibodies for binding tothe same epitope is determined by epitope binning. For example, labeledantigen is immobilized on a solid surface, and reacted with a saturatingamount of a first antibody. A second competing antibody is then added.Any additional binding by the second antibody as detected by, e.g.,OCTET® (ForteBio) or other bio-layer interferometry (BLI) techniques,indicates that the two antibodies bind to distinct, non-overlappingepitopes. No additional binding indicates that the two antibodies bindto the same or overlapping epitope. Several other methods, includingELISA, size exclusion chromotagraphy, crystallography, HDX-MS,mutagenesis, and other SPR methods, can also be employed to demonstratethat two antibodies bind to the same or overlapping epitopes. Similartechniques can be used to determine whether an antibody cross-blocks, oris cross-blocked by, the antibody of the invention.

2. Activity Assays

In one aspect, assays are provided for identifying anti-tryptaseantibodies thereof having biological activity. Biological activity mayinclude, for example, binding to tryptase (e.g., tryptase in thebloodstream or in the airway), or a peptide fragment thereof, either invivo, in vitro, or ex vivo. In other embodiments, biological activitymay include blocking or neutralizing tryptase. For example, in someembodiments, biological activity may include blocking or neutralizingtryptase proteolytic activity. Antibodies having such biologicalactivity in vivo and/or in vitro are also provided. In certainembodiments, an antibody of the invention is tested for such biologicalactivity.

For example, in some embodiments, an antibody of the invention is testedfor inhibition of tryptase activity in a tryptase enzymatic assay, forexample, using a peptide substrate (e.g., synthetic peptide S-2288), forexample, as described in the Examples (e.g., Example 1, particularlySection (A)(viii)(a)), or using methods known in the art (see, e.g.,Fukuoka et al. J. Immunol. 176:3165-3172, 2006). In some embodiments,inhibition of tryptase activity in a tryptase enzymatic assay isperformed at or around neutral pH (e.g., around pH 7, e.g., about pH 7,about pH. 7.4, or about pH 8). In other embodiments, inhibition oftryptase activity in a tryptase enzymatic assay is performed at anacidic pH (e.g., about pH 6.0, e.g., about pH 4.0, about pH 5.0, aboutpH 6.0, or about pH 6.5).

In other embodiments, an antibody of the invention is tested for theability to disrupt tryptase having a tetrameric structure (such asmature tryptase present in, or released from, mast cell secretorygranules) to form monomers, which may be determined using any suitablemethod known in the art, including gel filtration chromatography (e.g.,SUPEROSE®12 gel filtration chromatography). See, e.g., in Fukuoka et al.J. Immunol. 176:3165-3172,2006.

In yet other embodiments, an antibody of the invention is tested for theability to inhibit tryptase-stimulated proliferation and/or contractionof human primary airway smooth muscle cells, as described, for example,in Example 1.

In still other embodiments, an antibody of the invention is tested forthe ability to inhibit mast cell degranulation and/or histamine release,for example, stimulated by tryptase and/or IgE. Such assays aredescribed, for example, in Example 1.

In some embodiments, an antibody of the invention is tested for theability to inhibit active tryptase, for example, in an active tryptaseassay (see, e.g., FIG. 15 and Example 6), for example, in a sample(e.g., bronchoalveolar lavage fluid) obtained from a subject followingadministration of the antibody to the subject (e.g., by intravenous orsubcutaneous injection).

In still further embodiments, an antibody of the invention is tested forthe ability to modulate (e.g., increase or decrease) total tryptaselevels, for example, in a total tryptase assay (see, e.g., FIG. 15 andExample 6), for example, in a sample (e.g., bronchoalveolar lavagefluid) obtained from a subject following administration of the antibodyto the subject (e.g., by intravenous or subcutaneous injection).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-tryptaseantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al. Cancer Res. 53:3336-3342, 1993; and Lode et al.Cancer Res. 58:2925-2928, 1998); an anthracycline such daunomycin ordoxorubicin (see Kratz et al. Current Med. Chem. 13:477-523, 2006;Jeffrey et al. Bloorganic & Med. Chem. Letters 16:358-362, 2006; Torgovet al. Bioconj. Chem. 16:717-721, 2005; Nagy et al. Proc. Natl. Acad.Sci. USA 97:829-834, 2000; Dubowchik et al. Bioorg. & Med. Chem. Letters12:1529-1532, 2002; King et al. J. Med. Chem. 45:4336-4343, 2002; andU.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such asdocetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; atrichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example technetium-99m(tc99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238:1098, 1987.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (see, e.g., Chari et al. Cancer Res.52:127-131, 1992; U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-tryptase antibodies providedherein is useful for detecting the presence of tryptase in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, including but not limited to mast cells,basophils, epithelial cells, or lung tissue (e.g., bronchial smoothmuscle). In certain embodiments, a biological sample comprises blood(e.g., whole blood, serum, or plasma), sputum, bronchoalveolar lavage,or a nasosorption sample.

In one embodiment, an anti-tryptase antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of tryptase in a biological sample is provided.In certain embodiments, the method comprises contacting the biologicalsample with an anti-tryptase antibody as described herein underconditions permissive for binding of the anti-tryptase antibody totryptase, and detecting whether a complex is formed between theanti-tryptase antibody and tryptase. Such method may be an in vitro orin vivo method. In one embodiment, an anti-tryptase antibody is used toselect subjects eligible for therapy with an anti-tryptase antibody, forexample, where tryptase is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include pulmonary disorders (e.g., asthma (e.g., Th2-highasthma or Th2-ow asthma), airway hyperresponsiveness, or COPD),autoimmune disorders (e.g., rheumatoid arthritis, psoriasis,eosinophilic esophagitis, IBD, and Crohn's disease), inflammatorydisorders (e.g., chronic idiopathic urticaria (CIU or CSU), atopicdermatitis, or allergic rhinitis), fibrotic disorders (e.g., IPF),granulocytic (neutrophilic or eosinophilic) disorders, monocyticdisorders, lymphocytic disorders, disorders associated with increasednumbers or distribution of normal or aberrant tissue resident cells(such as mast cells, macrophages, or lymphocytes) or stromal cells (suchas fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia, and tryptase-associated disorders or tryptase-mediateddisorders.

For example, in some instances, the antibodies of the invention may beused to diagnose or detect anaphylaxis (e.g., acute systemicanaphylaxis) or mastocytosis (e.g., nonacute systemic mastocytosis), forexample, as described in Schwartz et al. Immunol. Allergy Clin. N. Am.26:451-463, 2006). The antibodies of the invention may be used, forexample, in an ELISA to detect the levels of total tryptase,pro-tryptase, and/or mature tryptase. In some embodiments, an antibodyof the invention may be used as a capture antibody in an ELISA. In otherembodiments, an antibody of the invention may be used as a detectionantibody in an ELISA. In particular embodiments, a normal referencelevel of total tryptase in serum or plasma may be about 1-15 ng/mL(e.g., about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL,about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14ng/mL, or about 15 ng/mL). In some instances, an increase relative to anormal reference level of total tryptase is used as a diagnosticindicator of a tryptase-associated disorder. In some embodiments, anormal reference level of mature tryptase in serum or plasma may be <1ng/mL. In some embodiments, an increase relative to a normal referencelevel of mature tryptase may be used as a diagnostic indicator of atryptase associated disorder.

In certain embodiments, labeled anti-tryptase antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luciferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, p-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-tryptase antibody of theinvention are prepared by mixing such antibody having the desired degreeof purity with one or more optional pharmaceutically acceptable carriers(see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed., 1980), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide aninterleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17)axis binding antagonist, an interleukin-5 (IL-5) axis bindingantagonist, an IL-33 axis binding antagonist, an M1 prime antagonist, anIgE antagonist, a TRPA1 antagonist, a CRTH2 antagonist, a broncodilatoror asthma symptom controller medication, an immunomodulator, acorticosteroid, a Th2 pathway inhibitor, a tyrosine kinase inhibitor, ora phosphodiesterase inhibitor. In some embodiments, the IL-13 axisbinding antagonist is an anti-IL-13 antibody, for example, lebrikizumab.In some embodiments, the IL-5 axis binding antagonist is an IL-5 bindingantagonist or an IL-5 receptor binding antagonist. In some embodiments,an IL-33 axis binding antagonist is an IL-33 binding antagonist or anST2 binding antagonist. In some embodiments, the IL-33 bindingantagonist is an anti-IL-33 antibody. In some instances, the M1 primeantagonist is quilizurnab. In some instances, the IgE antagonist isomalizumab (XOLAIR®). Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed., 1980.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, for example, byfiltration through sterile filtration membranes.

The invention provides pharmaceutical compositions that includeantioxidants. Such compositions may be used to reduce oxidation of anantibody described herein (e.g., an anti-tryptase antibody, such ashu31A.v11). In some instances, an antioxidant is included to reduceoxidation of a tryptophan residue, for example, a tryptophan residue ina HVR region or a FR region of a variable region. In particularinstances, an antioxidant is included to reduce oxidation of an HVR-H3residue of an anti-tryptase antibody, for example, W100 in hu31A.v1. Insome instances, an antioxidant is included in the composition to reduceoxidation of a methionine residue, for example a methionine residue inthe Fc region of an antibody (e.g., an anti-tryptase antibody), such asFc residues M251, M357, and/or M427 (for example, of hu31A.v1).

In some instances, the pharmaceutical composition includes any of theantibodies described herein and an antioxidant. In some instances, theantibody is susceptible to oxidation (e.g., at one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) tryptophan or methionine residues). Anysuitable antioxidant may be used. For example, in some instances, thecomposition includes one or more antioxidants (e.g., 1, 2, 3, 4, 5, 6,7, 8, or 9 antioxidants) selected from the group consisting ofN-acetyltryptophan (e.g., N-acetyl DL-tryptophan or N-acetylD-tryptophan), tryptophan, methionine (e.g., L-methionine), cysteine,glutathione, thiosorbitol, ascorbic acid, monothioglycerol,cyclodextrins, Trolox, pyridoxine, polyols (e.g., mannitol), sucrose, ametal chelator (e.g., EDTA), and combinations thereof (e.g., acombination of N-acetyltryptophan and methionine). In some instances,the antioxidant is N-acetyltryptophan (e.g., N-acetyl DL-tryptophan orN-acetyl D-tryptophan). In other instances, the antioxidant ismethionine (e.g., L-methionine or D-methionine). In particularinstances, the composition includes N-acetyltryptophan (e.g., N-acetylDL-tryptophan or N-acetyl D-tryptophan) and methionine (e.g.,L-methionine or D-methionine). In some instances, the N-acetyltryptophanreduces or prevents oxidation of tryptophan in the antibody. In someinstances, the methionine reduces or prevents oxidation of methionine inthe antibody.

The pharmaceutical composition may include any suitable concentration ofthe antioxidant in order to reduce or eliminate oxidation. For example,the concentration of polyols (e.g., mannitol) or sucrose may be about 1%(w/v) to about 25% (w/v), e.g., about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, or about 25% (w/v). In particular instances, theconcentration of polyols (e.g., mannitol) or sucrose may be about 15%(w/v). In another example, the concentration of metal chelators (e.g.,EDTA) may be about 0.01% (w/v) to about 1% (w/v), e.g., about 0.01%,about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%,about 0.25%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% (w/v). Inparticular instances, the concentration of a metal chelator (e.g., EDTA)may be about 0.4% (w/v). In yet another example, the concentration ofmethionine and/or tryptophan may be about 0.1 mg/ml to about 10 mg/ml,e.g., about 0.1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml,about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7mg/ml, about 8 mg/ml, about 9 mg/ml, or about 10 mg/ml. In someinstances, the concentration of methionine and/or tryptophan is about 2mg/ml.

For example, in some instances, the invention provides a pharmaceuticalcomposition that includes: (i) an isolated antibody that binds to humantryptase, or an antigen-binding fragment thereof, wherein the antibodycomprises the following six hypervariable regions (HVRs): (a) an HVR-H1comprising the amino acid sequence of DYGMV (SEQ ID NO: 7); (b) anHVR-H2 comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ IDNO: 2); (c) an HVR-H3 comprising the amino acid sequence of RNYDDWYFDV(SEQ ID NO: 8); (d) an HVR-L1 comprising the amino acid sequence ofSASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acidsequence of RTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising theamino acid sequence of QHYHSYPLT (SEQ ID NO: 6); (ii) N-acetyltryptophan(e.g., N-acetyl DL-tryptophan or N-acetyl D-tryptophan); and (iii)methionine (e.g., L-methionine or D-methionine).

Any suitable concentration of N-acetyltryptophan (e.g., N-acetylDL-tryptophan or N-acetyl D-tryptophan) may be used in any of thecompositions described herein. In some instances, the concentration ofN-acetyltryptophan (e.g., N-acetyl DL-tryptophan or N-acetylD-tryptophan) is about 0.05 mM to about 20 mM (e.g., about 0.05 mM toabout 20 mM, about 0.05 mM to about 19 mM, about 0.05 mM to about 18 mM,about 0.05 mM to about 17 mM, about 0.05 mM to about 16 mM, about 0.05mM to about 15 mM, about 0.05 mM to about 14 mM, about 0.05 mM to about13 mM, about 0.05 mM to about 13 mM, about 0.05 mM to about 12 mM, about0.05 mM to about 11 mM, about 0.05 mM to about 10 mM, about 0.05 mM toabout 9 mM, about 0.05 mM to about 8 mM, about 0.05 mM to about 7 mM,about 0.05 mM to about 6 mM, about 0.05 mM to about 5 mM, about 0.05 mMto about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM,about 0.05 mM to about 1 mM, about 0.1 mM to about 20 mM, about 0.1 mMto about 19 mM, about 0.1 mM to about 18 mM, about 0.1 mM to about 17mM, about 0.1 mM to about 16 mM, about 0.1 mM to about 15 mM, about 0.1mM to about 14 mM, about 0.1 mM to about 13 mM, about 0.1 mM to about 13mM, about 0.1 mM to about 12 mM, about 0.1 mM to about 11 mM, about 0.1mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to about 8mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mMto about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM,about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM toabout 0.9 mM, about 0.1 to about 0.8 mM, about 0.1 mM to about 0.7 mM,about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.5 mM, about 0.1 mMto about 0.4 mM, about 0.1 mM to about 0.3 mM, about 0.2 mM to about 20mM, about 0.2 mM to about 19 mM, about 0.2 mM to about 18 mM, about 0.2mM to about 17 mM, about 0.2 mM to about 16 mM, about 0.2 mM to about 15mM, about 0.2 mM to about 14 mM, about 0.2 mM to about 13 mM, about 0.2mM to about 13 mM, about 0.2 mM to about 12 mM, about 0.2 mM to about 11mM, about 0.2 mM to about 10 mM, about 0.2 mM to about 9 mM, about 0.2mM to about 8 mM, about 0.2 mM to about 7 mM, about 0.2 mM to about 6mM, about 0.2 mM to about 5 mM, about 0.2 mM to about 4 mM, about 0.2 mMto about 3 mM, about 0.2 mM to about 2 mM, about 0.2 mM to about 1 mM,about 0.2 mM to about 0.9 mM, about 0.2 to about 0.8 mM, about 0.2 mM toabout 0.7 mM, about 0.2 mM to about 0.6 mM, about 0.2 mM to about 0.5mM, about 0.2 mM to about 0.4 mM, about 0.2 mM to about 0.3 mM, about0.3 mM to about 20 mM, about 0.3 mM to about 19 mM, about 0.3 mM toabout 18 mM, about 0.3 mM to about 17 mM, about 0.3 mM to about 16 mM,about 0.3 mM to about 15 mM, about 0.3 mM to about 14 mM, about 0.3 mMto about 13 mM, about 0.3 mM to about 13 mM, about 0.3 mM to about 12mM, about 0.3 mM to about 11 mM, about 0.3 mM to about 10 mM, about 0.3mM to about 9 mM, about 0.3 mM to about 8 mM, about 0.3 mM to about 7mM, about 0.3 mM to about 6 mM, about 0.3 mM to about 5 mM, about 0.3 mMto about 4 mM, about 0.3 mM to about 3 mM, about 0.3 mM to about 2 mM,about 0.3 mM to about 1 mM, about 0.3 mM to about 0.9 mM, about 0.3 toabout 0.8 mM, about 0.3 mM to about 0.7 mM, about 0.3 mM to about 0.6mM, about 0.3 mM to about 0.5 mM, or about 0.3 mM to about 0.4 mM). Insome instances, the N-acetyltryptophan is at a concentration of about0.1 mM to about 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM,about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM,about 0.9 mM, or about 1 mM). In particular instances, the concentrationof N-acetyltryptophan (e.g., N-acetyl DL-tryptophan or N-acetylD-tryptophan) is about 0.3 mM. In particular instances, theN-acetyltryptophan is or N-acetyl DL-tryptophan.

Any suitable concentration of methionine (e.g., L-methionine orD-methionine) may be used in any of the compositions described herein.For example, in some instances, the concentration of methionine (e.g.,L-methionine or D-methionine) is about 0.1 mM to about 30 mM (e.g.,about 0.1 mM to about 30 mM, about 0.1 mM to about 25 mM, about 0.1 mMto about 20 mM, about 0.1 mM to about 19 mM, about 0.1 mM to about 18mM, about 0.1 mM to about 17 mM, about 0.1 mM to about 16 mM, about 0.1mM to about 15 mM, about 0.1 mM to about 14 mM, about 0.1 mM to about 13mM, about 0.1 mM to about 13 mM, about 0.1 mM to about 12 mM, about 0.1mM to about 11 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 7 mM, about 0.1 mMto about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM,about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM toabout 1 mM, about 1 mM to about 20 mM, about 1 mM to about 19 mM, about1 mM to about 18 mM, about 1 mM to about 17 mM, about 1 mM to about 16mM, about 1 mM to about 15 mM, about 1 mM to about 14 mM, about 1 mM toabout 13 mM, about 1 mM to about 13 mM, about 1 mM to about 12 mM, about1 mM to about 11 mM, about 1 mM to about 10 mM, about 1 mM to about 9mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM toabout 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 20 mM,about 2 mM to about 19 mM, about 2 mM to about 18 mM, about 2 mM toabout 17 mM, about 2 mM to about 16 mM, about 2 mM to about 15 mM, about2 mM to about 14 mM, about 2 mM to about 13 mM, about, 2 mM to about 13mM, about 2 mM to about 12 mM, about 2 mM to about 11 mM, about 2 mM toabout 10 mM, about 2 mM to about 9 mM, about 2 mM to about 8 mM, about 2mM to about 7 mM, about 2 mM to about 6 mM, about 2 mM to about 5 mM,about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 5 mM to about20 mM, about 5 mM to about 19 mM, about 5 mM to about 18 mM, about 5 mMto about 17 mM, about 5 mM to about 16 mM, about 5 mM to about 15 mM,about 5 mM to about 14 mM, about 5 mM to about 13 mM, about 5 mM toabout 13 mM, about 5 mM to about 12 mM, about 5 mM to about 11 mM, about5 mM to about 10 mM, about 5 mM to about 9 mM, about 5 mM to about 8 mM,about 5 mM to about 7 mM, or about 5 mM to about 6 mM). In someinstances, the methionine (e.g., L-methionine or D-methionine) is at aconcentration of about 1 mM to about 10 mM (e.g., about 1 mM, about 2mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8mM, about 9 mM, or about 10 mM). In particular instances, theconcentration of methionine (e.g., L-methionine or D-methionine) isabout 5 mM. In other instances, the concentration of methionine is about10 mM. In particular instances, the methionine is L-methionine.

In some instances, the presence of the antioxidant (e.g.,N-acetyltryptophan (e.g., N-acetyl DL-tryptophan) and/or methionine(e.g., L-methionine or D-methionine)) reduces the percent oxidation ofthe anti-tryptase antibody at a particular residue (e.g., a tryptophanresidue or a methionine residue, for example, in an HVR-H3 residue(e.g., W100 of the VH domain of hu31A.v1) or in an Fc residue (e.g., Fcresidues M251, M357, and/or M427 (for example, of hu31A.v1)) by about1%, about 2%, about 5%, about 6%, about 10%, about 15%, about 20%, about25%, about 28%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,or more, for example, relative to the percent oxidation at the residuein the absence of the antioxidant.

In instances where the antibody includes a VH domain W100 residue inHVR-H3 (e.g., hu31A.v11) that is susceptible to oxidation, in someinstances, the VH domain W100 residue in HVR-H3 has a percent oxidationof less than about 35% (e.g., less than 35%, less than 34%, less than33%, less than 32%, less than 31%, less than 30%, less than 29%, lessthan 28%, less than 27%, less than 26%, less than 25%, less than 24%,less than 23%, less than 22%, less than 21%, less than 20%, less than19%, less than 18%, less than 17%, less than 16%, less than 15%, lessthan 14%, less than 13%, less than 12%, less than 11%, less than 10%,less than 9%, less than 8%, less than 7%, less than 6%, less than 5%,less than 4%, less than 3%, less than 2%, less than 1%, or less). Forexample, in some instances, the W100 residue in HVR-H3 has a percentoxidation of less than about 35%. In other instances, the W100 residuein HVR-H3 has a percent oxidation of less than about 25%. In yet otherinstances, the W100 residue in HVR-H3 has a percent oxidation of lessthan about 15%. In still other instances, the W100 residue in HVR-H3 hasa percent oxidation of less than about 10%. In other instances, the W100residue in HVR-H3 has a percent oxidation of less than about 5%. Inother instances, the W100 residue in HVR-H3 has a percent oxidation ofless than about 4%, about 3%, about 2%, or about 1%.

As an example, in some instances where the antibody includes a VH domainW100 residue in HVR-H3 that is susceptible to oxidation, in someinstances, the VH domain W100 residue in HVR-H3 has a percent oxidationof between about 1% to about 50%, between about 1% to about 45%, betweenabout 1% to about 40%, between about 1% to about 35%, between about 1%to about 30%, between about 1% to about 25%, between about 1% to about20%, between about 1% to about 15%, between about 1% to about 10%,between about 1% to about 5%, between about 1% to about 4%, betweenabout 1% to about 3%, between about 1% to about 2%, between about 5% toabout 50%, between about 5% to about 45%, between about 5% to about 50%,between about 5% to about 35%, between about 5% to about 30%, betweenabout 5% to about 25%, between about 5% to about 20%, between about 5%to about 15%, between about 5% to about 10%, between about 10% to about50%, between about 10% to about 45%, between about 10% to about 40%,between about 10% to about 35%, between about 10% to about 30%, betweenabout 10% to about 25%, between about 10% to about 20%, between about10% to about 15%, between about 15% to about 50%, between about 15% toabout 45%, between about 15% to about 40%, between about 15% to about35%, between about 15% to about 30%, between about 15% to about 25%,between about 15% to about 20%, between about 25% to about 50%, orbetween about 30% to about 50%.

Percent oxidation may be determined, for example, within about 1 month,2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months 9months, 10 months, 11 months, 12 months, 18 months, two years, or morefrom the initial production of an antibody or a pharmaceuticalcomposition thereof. In some instances, percent oxidation is determinedabout 9 months, about 12 months, about 18 months, or two years from theinitial production of an antibody or a pharmaceutical compositionthereof.

The concentration of an antibody in a composition of the invention mayrange, for example, from about 1 mg/mL to about 350 mg/mL (e.g., about 1mg/mL to about 350 mg/mL, about 1 mg/mL to about 325 mg/mL, about 1mg/mL to about 300 mg/mL, about 1 mg/mL to about 275 mg/mL, about 1mg/mL to about 250 mg/mL, about 1 mg/mL to about 225 mg/mL, about 1mg/mL to about 200 mg/mL, about 1 mg/mL to about 175 mg/mL, about 1mg/mL to about 150 mg/mL, about 1 mg/mL to about 125 mg/mL, about 1mg/mL to about 100 mg/mL, about 1 mg/mL to about 75 mg/mL, about 1 mg/mLto about 50 mg/mL, about 1 mg/mL to about 25 mg/mL, about 25 mg/mL toabout 350 mg/mL, about 25 mg/mL to about 325 mg/mL, about 25 mg/mL toabout 300 mg/mL, about 25 mg/mL to about 275 mg/mL, about 25 mg/mL toabout 250 mg/mL, about 25 mg/mL to about 225 mg/mL, about 25 mg/mL toabout 200 mg/mL, about 25 mg/mL to about 175 mg/mL, about 25 mg/mL toabout 150 mg/mL, about 25 mg/mL to about 125 mg/mL, about 25 mg/mL toabout 100 mg/mL, about 25 mg/mL to about 75 mg/mL, about 25 mg/mL toabout 50 mg/mL, about 50 mg/mL to about 350 mg/mL, about 50 mg/mL toabout 325 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL toabout 275 mg/mL, about 50 mg/mL to about 250 mg/mL, about 50 mg/mL toabout 225 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL toabout 175 mg/mL, about 50 mg/mL to about 150 mg/mL, about 50 mg/mL toabout 125 mg/mL, about 50 mg/mL to about 100 mg/mL, about 50 mg/mL toabout 75 mg/mL, about 75 mg/mL to about 350 mg/mL, about 75 mg/mL toabout 325 mg/mL, about 75 mg/mL to about 300 mg/mL, about 75 mg/mL toabout 275 mg/mL, about 75 mg/mL to about 250 mg/mL, about 75 mg/mL toabout 225 mg/mL, about 75 mg/mL to about 200 mg/mL, about 75 mg/mL toabout 175 mg/mL, about 75 mg/mL to about 150 mg/mL, about 75 mg/mL toabout 125 mg/mL, about 75 mg/mL to about 100 mg/mL, about 100 mg/mL toabout 350 mg/mL, about 100 mg/mL to about 325 mg/mL, about 100 mg/mL toabout 300 mg/mL, about 100 mg/mL to about 275 mg/mL, about 100 mg/mL toabout 250 mg/mL, about 100 mg/mL to about 225 mg/mL, about 100 mg/mL toabout 200 mg/mL, about 100 mg/mL to about 175 mg/mL, about 100 mg/mL toabout 150 mg/mL, about 100 mg/mL to about 125 mg/mL, or about 150 mg/mLto about 175 mg/mL. In some instances, the antibody is at aconcentration of about 50 mg/mL to about 200 mg/mL (e.g., about 50mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL,about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL,about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about 170 mg/mL,about 180 mg/mL, about 190 mg/mL, or about 200 mg/mL. In particularinstances, the antibody concentration is about 150 mg/mL.

Any of the compositions described herein may include one or moreadditional excipients selected from the group consisting of astabilizer, a buffer, a surfactant, and a tonicity agent. In someinstances, the buffer comprises arginine succinate and/or histidinesuccinate. In some instances, the buffer includes arginine succinate andhistidine succinate.

Any suitable concentration of arginine succinate may be used. Forexample, in some instances, the concentration of arginine succinate isabout 10 mM to about 500 mM (e.g., about 10 mM, about 20 mM, about 30mM, about 40 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM,about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM,about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM,about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500mM). For example, in some instances, the concentration of argininesuccinate is about 100 mM to about 300 mM, about 125 mM to about 300 mM,about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mMto about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300mM, about 275 mM to about 300 mM, about 100 mM to about 250 mM, about125 mM to about 250 mM, about 150 mM to about 250 mM, about 175 mM toabout 250 mM, about 200 mM to about 250 mM, about 100 mM to about 225mM, about 125 mM to about 225 mM, about 150 mM to about 225 mM, about175 mM to about 225 mM, about 200 mM to about 225 mM, about 100 mM toabout 200 mM, about 125 mM to about 200 mM, about 150 mM to about 200mM, or about 175 mM to about 200 mM. In particular instances, theconcentration of arginine succinate is about 200 mM.

Any suitable concentration of histidine succinate may be used. Forexample, in some embodiments, the concentration of histidine succinateis about 1 mM to about 100 mM (e.g., about 1 mM, about 5 mM, about 10mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM,about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM,about 95 mM, or about 100 mM). In particular instances, theconcentration of histidine succinate is about 20 mM.

Any of the compositions described herein may have a pH of about 4.0 toabout 7.0 (e.g., about 4.0, about 4.5, about 5.0, about 5.5, about 6.0,about 6.5, or about 7.0). In some instances, the pH is from about 4.5 toabout 7.0 (e.g., about 4.5, about 5.0, about 5.5, about 6.0, about 6.5,or about 7.0). In some instances, the pH is from about 4.5 to about 6.5(e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5,about 5.1, about 5.2, about 5.3, about 5.4, about 5.5 about 5.6, about5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3,about 6.4, or about 6.5). In some instances, the pH is from about 5.0 toabout 6.0 (e.g., about 5.0, about 5.1, about 5.2, about 5.3, about 5.4,about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0). Insome instances, the pH is about 5.5.

Any suitable surfactant may be used in the compositions describedherein. In some instances, the surfactant is poloxamer 188 orpolysorbate 20. Any suitable concentration of poloxamer 188 may be used.For example, the concentration of poloxamer 188 may be about 0.005% toabout 0.5%, about 0.005% to about 0.05%, or about 0.02%. In someinstances, the concentration of poloxamer 188 is about 0.01%, about0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%,about 0.08%, about 0.09%, or about 0.1%. In particular embodiments, theconcentration of poloxamer 188 is about 0.02%. Any suitableconcentration of polysorbate 20 may be used. For example, theconcentration of polysorbate 20 may be about 0.005% to about 0.5%, about0.005% to about 0.05%, or about 0.02%. In some instances, theconcentration of polysorbate 20 is about 0.01%, about 0.02%, about0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%,about 0.09%, or about 0.1%.

In particular instances, the composition includes about 150 mg/mL of ananti-tryptase antibody described herein (e.g., hu31A.v11), about 200 mMarginine succinate, about 20 mM histidine succinate, about 0.3 mMN-acetyl DL-tryptophan), about 5 mM L-methionine, about 0.02% poloxamer188, and has a pH of about 5.8.

Any of the compositions described herein may be formulated foradministration by any suitable route. In particular instances, thecomposition is formulated for subcutaneous administration or intravenousadministration. In some instances, the composition is formulated forsubcutaneous administration.

G. Therapeutic Methods and Compositions

Any of the anti-tryptase antibodies or pharmaceutical compositions ofthe invention may be used in therapeutic methods.

In one aspect, an anti-tryptase antibody, or a pharmaceuticalcomposition thereof, for use as a medicament is provided. In furtheraspects, an anti-tryptase antibody, or a pharmaceutical compositionthereof, for use in treating a disorder is provided. In certainembodiments, an anti-tryptase antibody, or a pharmaceutical compositionthereof, for use in a method of treatment is provided. In certainembodiments, the invention provides an anti-tryptase antibody, or apharmaceutical composition thereof, for use in a method of treating asubject having a disorder that involves administering to the subject aneffective amount of the anti-tryptase antibody. In some embodiments, themethod further includes administering to the subject an effective amountof at least one additional therapeutic agent, for example, as describedbelow. A “subject” according to any of the above embodiments ispreferably a human.

In a further aspect, the invention provides for the use of ananti-tryptase antibody, or a pharmaceutical composition thereof, in themanufacture or preparation of a medicament. In one embodiment, themedicament is for treatment of a disorder. In a further embodiment, themedicament is for use in a method of treating a disorder comprisingadministering to a subject having the disorder an effective amount ofthe medicament. In one such embodiment, the method further comprisesadministering to the subject an effective amount of at least oneadditional therapeutic agent, e.g., as described below. A “subject”according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating adisorder, including, for example, a pulmonary disorder (e.g., asthma(e.g., Th2-high asthma or Th2-low asthma), airway hyperresponsiveness,or COPD), an autoimmune disorder (e.g., rheumatoid arthritis, psoriasis,eosinophilic esophagitis, IBD, or Crohn's disease), an inflammatorydisorder (e.g., chronic idiopathic urticaria (CIU or CSU), anaphylaxis,atopic dermatitis, or allergic rhinitis), a fibrotic disorder (e.g.,IPF), a granulocytic (neutrophilic or eosinophilic) disorder, amonocytic disorder, a lymphocytic disorder, a disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia, and a tryptase-associated disorder or tryptase-mediateddisorder. In one embodiment, the method comprises administering to asubject having a disorder an effective amount of an anti-tryptaseantibody, or a pharmaceutical composition thereof. In one suchembodiment, the method further comprises administering to the subject aneffective amount of at least one additional therapeutic agent, asdescribed below. A “subject” according to any of the above embodimentsmay be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-tryptase antibodies provided herein, e.g.,for use in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-tryptase antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of theanti-tryptase antibodies provided herein and at least one additionaltherapeutic agent, for example, as described below. Any of thepharmaceutical formulations described in Section F (e.g., those thatinclude an antioxidant such as N-acetyltryptophan and/or methionine)above may be used in any of the uses or methods described herein.

In some embodiments, the disorder is an autoimmune disorder, aninflammatory disorder, a fibrotic disorder, a granulocytic (neutrophilicor eosinophilic) disorder, a monocytic disorder, a lymphocytic disorder,or a disorder associated with increased numbers or distribution ofnormal or aberrant tissue resident cells (such as mast cells,macrophages, or lymphocytes) or stromal cells (such as fibroblasts,myofibroblasts, smooth muscle cells, epithelia, or endothelia). In someembodiments, the disorder is a pulmonary disorder.

In some embodiments, the pulmonary disorder is associated withgranulocytic (eosinophilic and/or neutrophilic) pulmonary inflammation,infection-induced pulmonary conditions (including those associated withviral (e.g., influenza, parainfluenza, rhinovirus, humanmetapneumovirus, and respiratory syncytial virus), bacterial, or fungal(e.g., Aspergilius) triggers. In some embodiments, the disorder is anallergen-induced pulmonary condition, a toxic environmentalpollutant-induced pulmonary condition (e.g., asbestosis, silicosis, orberylliosis), a gastric aspiration-induced pulmonary condition, orassociated with immune dysregulation or an inflammatory condition withgenetic predisposition such as cystic fibrosis. In some embodiments, thedisorder is a physical trauma-induced pulmonary condition (e.g.,ventilator injury), emphysema, cigarette-induced emphysema, bronchitis,sarcoidosis, histiocytosis, lymphangiomyomatosis, acute lung injury,acute respiratory distress syndrome, chronic lung disease,bronchopulmonary dysplasia, pneumonia (e.g., community-acquiredpneumonia, nosocomial pneumonia, ventilator-associated pneumonia, viralpneumonia, bacterial pneumonia, and severe pneumonia), airwayexacerbations, and acute respiratory distress syndrome (ARDS)). In someembodiments, the pulmonary disorder is COPD.

In some embodiments of any of the methods, the pulmonary disorder isasthma. In some embodiments, the asthma is persistent chronic severeasthma with acute events of worsening symptoms (exacerbations or flares)that can be life threatening. In some embodiments, the asthma is atopic(also known as allergic) asthma, non-allergic asthma (e.g., oftentriggered by infection with a respiratory virus (e.g., influenza,parainfluenza, rhinovirus, human metapneumovirus, and respiratorysyncytial virus) or inhaled irritant (air pollutants, smog, dieselparticles, volatile chemicals and gases indoors or outdoors, or even bycold dry air).

In some embodiments of any of the methods, the asthma is intermittent orexercise-induced, asthma due to acute or chronic primary or second-handexposure to “smoke” (typically cigarettes, cigars, pipes), inhaling or“vaping” (tobacco, marijuana or other such substances), or asthmatriggered by recent ingestion of aspirin or related NSAIDS. In someembodiments, the asthma is mild, or corticosteroid naïve asthma, newlydiagnosed and untreated asthma, or not previously requiring chronic useof inhaled topical or systemic steroids to control the symptoms (cough,wheeze, shortness of breath/breathlessness, or chest pain). In someembodiments, the asthma is chronic, corticosteroid resistant asthma,corticosteroid refractory asthma, asthma uncontrolled on corticosteroidsor other chronic asthma controller medications.

In some embodiments of any of the methods, the asthma is moderate tosevere asthma. In certain embodiments, the asthma is Th2-high asthma. Insome embodiments, the asthma is severe asthma. In some embodiments, theasthma is atopic asthma, allergic asthma, non-allergic asthma (e.g., dueto infection and/or respiratory syncytial virus (RSV)), exercise-inducedasthma, aspirin sensitive/exacerbated asthma, mild asthma, moderate tosevere asthma, corticosteroid naïve asthma, chronic asthma,corticosteroid resistant asthma, corticosteroid refractory asthma, newlydiagnosed and untreated asthma, asthma due to smoking, asthmauncontrolled on corticosteroids. In some embodiments, the asthma is Thelper lymphocyte type 2 (Th2) or type 2 (Th2) high, or Type 2(T2)-driven asthma. In some embodiments, the asthma is eosinophilicasthma. In some embodiments, the asthma is allergic asthma. In someembodiments, the individual has been determined to be EosinophilicInflammation Positive (EIP). See WO2015/061441. In some embodiments, theasthma is periostin-high asthma (e.g., having periostin level at leastabout any of 20 ng/mL, 25 ng/mL, or 50 ng/mL serum). Methods ofdetermining serum periostin levels are provided, for example, inUS2012/0156194. In some embodiments, the asthma is eosinophil-highasthma (e.g., at least about any of 150, 200, 250, 300, 350, 400eosinophil counts/ml blood). In certain embodiments, the asthma isTh2-low asthma or nonTh2-driven asthma. In some embodiments, theindividual has been determined to be Eosinophilic Inflammation Negative(EIN). See WO2015/061441. In some embodiments, the asthma isperiostin-low asthma (e.g., having periostin level less than about 20ng/mL serum). In some embodiments, the asthma is eosinophil-low asthma(e.g., less than about 150 eosinophil counts/μl blood or less than about100 eosinophil counts/μl blood). In certain embodiments, the individualexhibits elevated level of FeNO (fractional exhaled nitric acid) and/orelevated level of IgE. For example, in some instances, the individualexhibits a FeNO level above about 250 parts per billion (ppb), aboveabout 275 ppb, above about 300 ppb, above about 325 ppb, above about 325ppb, or above about 350 ppb. In some instances, the individual has anIgE level that is above 50 IU/ml. In some embodiments, the individualhas a forced expiratory volume in 1 second (FEV1) of 40% to 80% ofpredicted.

In other embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, neutrophilic disorder, oreosinophilic disorder is pulmonary fibrosis. In some embodiments, thepulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).

In still further embodiments of any of the methods, the autoimmunedisorder, inflammatory disorder, fibrotic disorder, granulocytic(neutrophilic or eosinophilic) disorder, monocytic disorder, orlymphocytic disorder is esophagitis (e.g., eosinophilic esophagitis),allergic rhinitis, non-allergic rhinitis, rhinosinusitis with polyps,nasal polyposis, bronchitis, chronic pneumonia, allergicbronchopulmonary aspergillosis, airway inflammation, allergic rhinitis,bronchiectasis, and/or chronic bronchitis.

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic oreosinophilic) disorder, monocytic disorder, or lymphocytic disorder, isarthritis. In some embodiments, the arthritis is rheumatoid arthritis.In some embodiments, the arthritis is osteoarthritis, rheumatoidarthritis, juvenile arthritis, juvenile rheumatoid arthritis, earlyarthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoidarthritis, enteropathic arthritis, reactive arthritis, psoriaticarthritis, and/or arthritis as a result of injury.

In yet other embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic oreosinophilic) disorder, monocytic disorder, or lymphocytic disorder is agastrointestinal inflammatory condition. In some embodiments, thegastrointestinal inflammatory condition is IBD (inflammatory boweldisease), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g.,colitis caused by environmental insults (e.g., caused by or associatedwith a therapeutic regimen, such as chemotherapy, radiation therapy,etc.)), infectious colitis, ischemic colitis, collagenous or lymphocyticcolitis, necrotizing enterocolitis, colitis in conditions such aschronic granulomatous disease or celiac disease, food allergies,gastritis, gastroenteritis, infectious gastritis or enterocolitis (e.g.,Helicobacter pylori-infected chronic active gastritis), esophagitis, andother forms of gastrointestinal inflammation caused by an infectiousagent, or indeterminate colitis.

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic oreosinophilic) disorder, monocytic disorder, or lymphocytic disorder is agastrointestinal inflammatory condition. In some embodiments, thegastrointestinal inflammatory condition is IBD (inflammatory boweldisease). In some embodiments the inflammatory bowel disease isulcerative colitis (UC) or Crohn's disease (CD). In some embodiments,the gastrointestinal inflammatory condition is colitis (e.g., colitiscaused by environmental insults (e.g., caused by or associated with atherapeutic regimen, such as chemotherapy, radiation therapy, etc.),infectious colitis, ischemic colitis, collagenous or lymphocyticcolitis, necrotizing enterocolitis, colitis in conditions such aschronic granulomatous disease or celiac disease, food allergies,gastritis, gastroenteritis, infectious gastritis or enterocolitis (e.g.,Helicobacter pylori-infected chronic active gastritis), and other formsof gastrointestinal inflammation caused by an infectious agent, orindeterminate colitis.

In some embodiments of any of the methods, the gastrointestinalinflammatory condition is ulcerative colitis (UC) or Crohn's disease(CD). In some embodiments, the gastrointestinal inflammatory conditionis ulcerative colitis (UC). In some embodiments, the ulcerative colitisis mild to moderate distal colitis. In some embodiments, the ulcerativecolitis is mild to moderate extensive colitis. In some embodiments, theulcerative colitis is severe colitis. In some embodiments, thegastrointestinal inflammatory condition is Crohn's disease (CD). In someembodiments, the Crohn's disease is in acute disease stage. In someembodiments, the Crohn's disease is in induced clinical remission stage.In some embodiments, the Crohn's disease is in maintainresponse/remission stage. In some embodiments, the Crohn's disease ismild to moderate disease. In some embodiments, the Crohn's disease ismoderate to severe disease. In some embodiments, the Crohn's disease issevere/fulminant disease. In some embodiments, the Crohn's disease isileal, ileocolonic, or colonic disease.

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic oreosinophilic) disorder, monocytic disorder, or lymphocytic disorder, ordisorder associated with increased numbers or distribution of normal oraberrant tissue resident cells (such as mast cells, macrophages, orlymphocytes) or stromal cells (such as fibroblasts, myofibroblasts,smooth muscle cells, epithelia, or endothelia) is lupus or SystemicLupus Erythematosus (SLE), or one or more organ-specific manifestationsof lupus (e.g., lupus nephritis (LN) affecting the kidney, orextra-renal lupus (ERL) affecting the blood and/or lymphoid organs(lymph nodes, spleen, thymus, and associated lymphatic vessels), and/orjoints and/or other organs, but not necessarily the kidney).

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, or fibrotic disorder is related to sepsis and/ortrauma, HIV infection, or idiopathic (of unknown etiology) such asANCA-associated vaculitides (AAV), granulomatosis with polyangiitis(formerly known as Wegener's granulomatosis), Behcet's disease,cardiovascular disease, eosinophilic bronchitis, Reiter's Syndrome, SEASyndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),ankylosing spondylitis, dermatomyositis, scleroderma, e.g., systemicscleroderma also called systemic sclerosis, vasculitis (e.g., Giant CellArteritis (GCA), also called temporal arteritis, cranial arteritis orHorton disease), myositis, polymyositis, dermatomyositis, polyarteritisnodosa, arteritis, polymyalgia rheumatica, sarcoidosis, primary biliarysclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaquepsoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis,erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus, e.g.,pemphigus vulgaris, atherosclerosis, lupus, Still's disease, myastheniagravis, celiac disease, multiple sclerosis (MS) of therelapsing-remitting (RRMS) or primary progressive (PPMS) or secondaryprogressive (SPMS) subtypes, Guillain-Barre disease, Type I diabetesmellitus (T1DM) or insulin-dependent (IDDM) or juvenile onset DM type,thyroiditis (e.g., Graves' disease), coeliac disease, Churg-Strausssyndrome, myalgia syndrome, hypereosinophilic syndrome, oedematousreactions including episodic angioedema, helminth infections,onchocercal dermatitis, eosinophilic esophagitis, eosinophilicenteritis, eosinophilic colitis, obstructive sleep apnea, endomyocardialfibrosis, Addison's disease, Raynaud's disease or phenomenon, autoimmunehepatitis, graft versus host disease (GVHD), or organ transplantrejection.

In some embodiments of any of the methods, the disorder is aninflammatory disorder of the skin. In some embodiments, the disorder isatopic dermatitis or onchocercal dermatitis. In some embodiments, thedisorder is chronic idiopathic urticaria (CIU or CSU). In someembodiments of any of the methods, the method further comprisesadministering an IL-13 antagonist, an IL-33 antagonist, an IgEantagonist, a calcium inhibitor, a leukotrieine inhibitor, or anantihistamine. In some embodiments, the IL-13 antagonist islebrikizumab. In some embodiments, the IgE antagonist is omalizumab orligelizumab.

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, neutrophilic disorder, oreosinophilic disorder is a fibrotic disorder. In some embodiments, thefibrotic disorders include lung fibrosis, liver fibrosis (e.g., fibrosisassociated with cirrhosis (e.g., alcohol-induced cirrhosis,viral-induced cirrhosis, post-hepatitis C cirrhosis, and primary biliarycirrhosis), schistosomiasis, cholangitis (e.g., sclerosing cholangitis),and autoimmune-induced hepatitis), kidney fibrosis (e.g.,tubulointerstitial fibrosis, scleroderma, diabetic nephritis, andglomerular nephritis), dermal fibrosis (e.g., scleroderma, hypertrophicand keloid scarring, nephrogenic fibrosing dermatopathy, and burns),myelofibrosis, neurofibromatosis, fibroma, intestinal fibrosis, andfibrotic adhesions resulting from surgical procedures), heart fibrosis(e.g., fibrosis associated with myocardial infarction), vascularfibrosis (e.g., fibrosis associated with postangioplasty arterialrestenosis and atherosclerosis), eye fibrosis (e.g., fibrosis associatedwith post-cataract surgery, proliferative vitreoretinopathy, andretro-orbital fibrosis), and bone marrow fibrosis (e.g., idiopathicmyelofibrosis and drug-induced myelofibrosis). The fibrosis can beorgan-specific or systemic (e.g., systemic sclerosis and fibrosisassociated with GVHD). In some embodiments, the fibrotic disorder ispulmonary fibrosis. In some embodiments, the pulmonary fibrosis isfibrosing interstitial pneumonia. In some embodiments, the pulmonaryfibrosis is idiopathic pulmonary fibrosis (IPF), also known ascryptogenic fibrosing alveolitis. In some embodiments, the IPF isgender, age and physiology (GAP)-stage I. In some embodiments, the IPFis GAP-stage II. In some embodiments, the IPF is GAP-stage III. In someembodiments, the pulmonary fibrosis is sporadic IPF. In someembodiments, the pulmonary fibrosis is familial pulmonary fibrosis. Insome embodiments, the pulmonary fibrosis is combined pulmonary fibrosisand emphysema. In some embodiments, the pulmonary fibrosis is associatedwith one or more of the following: usual interstitial pneumonia;idiopathic interstitial pneumonia; desquamative interstitial pneumonia;respiratory bronchiolitis-interstitial lung disease; acute interstitialpneumonia; nonspecific interstitial pneumonia; sarcoidosis; cryptogenicorganizing pneumonia; eosinophilic pneumonia; infection; exposure tooccupational or environmental agents; cigarette smoking; interstitiallung disease induced by drugs or radiation; rheumatic disease-associatedinterstitial lung disease; lymphoid interstitial pneumonia;pleuropulmonary fibroelastosis; pulmonary Langerhans cell histiocytosis;systemic sclerosis-interstitial lung disease; Hermansky-Pudlak syndrome;and telomeropathy.

In some embodiments of any of the methods, the autoimmune disorder,inflammatory disorder, fibrotic disorder, neutrophilic disorder, oreosinophilic disorder is chronic obstructive pulmonary disease (COPD).In some embodiments, the COPD is Global Initiative for ChronicObstructive Lung Disease (GOLD) category A. In some embodiments, theCOPD is GOLD category B. In some embodiments, the COPD is GOLD categoryC. In some embodiments, the COPD is GOLD category D. In someembodiments, the COPD is chronic bronchitis. In some embodiments, theCOPD is emphysema. In some embodiments, the emphysema is proximalacinar, panacinar, or distal acinar emphysema. In some embodiments, theemphysema is cigarette-induced emphysema. In some embodiments, the COPDis associated with exposure to particulate dusts, chemical fumes, and/orair pollution. In some embodiments, the COPD is associated with impairedlung development. In some embodiments, the COPD is chronic obstructiveasthma. In some embodiments, the COPD is associated with alpha-1antitrypsin deficiency. In some embodiments, the COPD is associated withserine protease inhibitor clade E, member 2 (SERPINE2) disruption. Insome embodiments, the COPD is COPD with persistent systemicinflammation. In some embodiments, the COPD is eosinophilic or T-helpertype 2 (T_(H)2) high COPD. In some embodiments, the COPD is COPD withpersistent bacterial colonization. In some embodiments, the COPD is COPDwith frequent exacerbations. In some embodiments, the autoimmunedisorder, inflammatory disorder, fibrotic disorder, neutrophilicdisorder, or eosinophilic disorder is asthma-COPD overlap syndrome(ACOS). In some embodiments, the ACOS is eosinophil-predominant,neutrophil-predominant, mixed-pattern, or no inflammation(paucigranulocytic) ACOS. In some embodiments, the autoimmune disorder,inflammatory disorder, fibrotic disorder, a neutrophilic disorder, or aneosinophilic disorder is COPD-obstructive sleep apnea (OSA) overlapsyndrome.

Antibodies of the invention, or pharmaceutical compositions thereof, canbe used either alone or in combination with other agents in a therapy.For instance, an antibody of the invention, or a pharmaceuticalcompositions thereof, may be co-administered with at least oneadditional therapeutic agent. In certain embodiments, an additionaltherapeutic agent is an interleukin-13 (IL-13) binding antagonist, aninterleukin-17 (IL-17) axis binding antagonist, an interleukin-5 (IL-5)axis binding antagonist, or an IL-33 axis binding antagonist. In someembodiments, the IL-13 axis binding antagonist is an anti-IL-13antibody, for example, lebrikizumab. In some embodiments, the IL-5 axisbinding antagonist is an IL-5 binding antagonist or an IL-5 receptorbinding antagonist. In some embodiments, an IL-33 axis bindingantagonist is an IL-33 binding antagonist or an ST2 binding antagonist.In some embodiments, the IL-33 binding antagonist is an anti-IL-33antibody.

In some embodiments, an additional therapeutic agent is an asthmatherapy, as described below. Moderate asthma is currently treated with adaily inhaled anti-inflammatory-corticosteroid or mast cell inhibitorsuch as cromolyn sodium or nedocromil plus an inhaled beta2-agonist asneeded (3-4 times per day) to relieve breakthrough symptoms or allergen-or exercise-induced asthma. Exemplary inhaled corticosteroids includeQVAR®, PULMICORT®, SYMBICORT®, AEROBID®, FLOVENT®, FLONASE®, ADVAIR®,and AZMACORT®. Additional asthma therapies include long acting bronchialdilators (LABD). In certain embodiments, the LABD is a long-actingbeta-2 agonist (LABA), leukotriene receptor antagonist (LTRA),long-acting muscarinic antagonist (LAMA), theophylline, or oralcorticosteroids (OCS). Exemplary LABDs include SYMBICORT®, ADVAIR®,BROVANA®, FORADIL®, PERFOROMIST™, and SEREVENT®.

In some embodiments of any of the methods, the method further comprisesadministering a bronchodilator or asthma symptom controller medication.In some embodiments, the bronchodilator or asthma controller medicationis a β2-adrenergic agonist, such as a short-acting 32-agonist (SABA)(such as albuterol), or a long-acting β2-adrenergic agonist (LABA). Insome embodiments, the LABA is salmeterol, abediterol, indacaterol,vilanterol, and/or formoterol (formoterol fumarate dehydrate). In someembodiments, the asthma controller medication is a Leukotriene ReceptorAntagonist (LTRA). In some embodiments, the LTRA is montelukast,zafirlukast, and/or zileuton. In some embodiments, the bronchodilator orasthma controller medication is a muscarinic antagonist, such as along-acting muscarinic acetylcholine receptor (cholinergic) antagonist(LAMA). In some embodiments, the LAMA is glycopyrronium. In someembodiments, the bronchodilator or asthma controller medication is anagonist of an ion channel such as a bitter taste receptor (such asTAS2R).

In some embodiments of any of the methods, the method further comprisesadministering a bronchodilator. In some embodiments, the bronchodilatoris an inhaled bronchodilator. In some embodiments, the inhaledbronchodilator is a 32-adrenergic agonist. In some embodiments, the32-adrenergic agonist is a short-acting 32-adrenergic agonist (SABA). Insome embodiments, the SABA is bitolterol, fenoterol, isoproterenol,levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine,albuterol, and/or terbutaline. In some embodiments, the 32-adrenergicagonist is a long-acting 32-adrenergic agonist (LABA). In someembodiments, the LABA is arformoterol, bambuterol, clenbuterol,formoterol, salmeterol, abediterol, carmoterol, indacaterol, olodaterol,and/or vilanterol. In some embodiments, the inhaled bronchodilator is amuscarinic receptor antagonist. In some embodiments, the muscarinicreceptor antagonist is a short-acting muscarinic receptor antagonist(SAMA). In some embodiments, the SAMA is ipratropium bromide. In someembodiments, the muscarinic receptor antagonist is a long-actingmuscarinic receptor antagonist (LAMA). In some embodiments, the LAMA istiotropium bromide, glycopyrronium bromide, umeclidinium bromide,aclidinium bromide, and/or revefenacin. In some embodiments, the inhaledbronchodilator is a SABA/SAMA combination. In some embodiments,SABA/SAMA combination is albuterol/ipratropium. In some embodiments, theinhaled bronchodilator is a LABA/LAMA combination. In some embodiments,the LABA/LAMA combination is formoterol/aclidinium,formoterol/glycopyrronium, formoterol/tiotropium,indacaterol/glycopyrronium, indacaterol/tiotropium,olodaterol/tiotropium, salmeterol/tiotropium, and/orvilanterol/umeclidinium. In some embodiments, the inhaled bronchodilatoris a bifunctional bronchodilator. In some embodiments, the bifunctionalbronchodilator is a muscarinic antagonist/β2-agonist (MABA). In someembodiments, the MABA is batefenterol, THRX 200495, AZD 2115, LAS190792, TE13252, PF-3429281 and/or PF-4348235. In some embodiments, theinhaled bronchodilator is an agonist of TAS2R. In some embodiments, thebronchodilator is a nebulized SABA. In some embodiments, the nebulizedSABA is albuterol and/or levalbuterol. In some embodiments, thebronchodilator is a nebulized LABA. In some embodiments, the nebulizedLABA is arformoterol and/or formoterol. In some embodiments, thebronchodilator is a nebulized SAMA. In some embodiments, the nebulizedSAMA is ipratropium. In some embodiments, the bronchodilator is anebulized LAMA. In some embodiments, the nebulized LAMA isglycopyrronium and/or revefenacin. In some embodiments, thebronchodilator is a nebulized SABA/SAMA combination. In someembodiments, the nebulized SABA/SAMA combination isalbuterol/ipratropium. In some embodiments, the bronchodilator is aleukotriene receptor antagonist (LTRA). In some embodiments, the LTRA ismontelukast, zafirlukast, and/or zileuton. In some embodiments, thebronchodilator is a methylxanthine. In some embodiments, themethylxanthine is theophylline.

In some embodiments of any of the methods, the method further comprisesadministering an immunomodulator. In some embodiments, theimmunomodulator is an antibody to immunoglobulin type E (IgE) oranti-IgE (an IgE inhibitor). In some embodiments, the anti-IgE isomalizumab and/or ligelizumab. In some embodiments of any of themethods, the method further comprises cromolyn. In some embodiments ofany of the methods, the method further comprises methylxanthine. In someembodiments, the methylxanthine is theophylline or caffeine.

In some embodiments of any of the methods, the method further comprisesadministering one or more corticosteroids, such as an inhaledcorticosteroid (ICS) or an oral corticosteroid. Non-limiting exemplarycorticosteroids include inhaled corticosteroids, such as beclomethasonedipropionate, budesonide, ciclesonide, flunisolide, fluticasonepropionate, fluticasone furoate, mometasone, and/or triamcinoloneacetonide and oral corticosteroids, such as methylprednisolone,prednisolone, and prednisone. In some embodiments, the corticosteroid isan ICS. In some embodiments, the ICS is beclomethasone, budesonide,flunisolide, fluticasone furoate, fluticasone propionate, mometasone,ciclesonide, and/or triamcinolone. In some embodiments of any of themethods, the method further comprises administering an ICS/LABA and/orLAMA combination. In some embodiments, the ICS/LABA and/or LAMAcombination is fluticasone propionate/salmeterol, budesonide/formoterol,mometasone/formoterol, fluticasone furoate/vilanterol, fluticasonepropionate/formoterol, beclomethasone/formoterol, fluticasonefuroate/umeclidinium, fluticasone furoate/vilanterol/umeclidinium,fluticasone/salmeterol/tiotropium,beclomethasone/formoterol/glycopyrronium,budesonide/formoterol/glycopyrronium, and/orbudesonide/formoterol/tiotropium. In some embodiments of any of themethods, the method further comprises administering a nebulizedcorticosteroid. In some embodiments, the nebulized corticosteroid isbudesonide. In some embodiments of any of the methods, the methodfurther comprises administering an oral or intravenous corticosteroid.In some embodiments, the oral or intravenous corticosteroid isprednisone, prednisolone, methylprednisolone, and/or hydrocortisone

In some embodiments of any of the methods, the method further comprisesadministering a TH2 or T2 pathway inhibitor that inhibits one or moretargets selected from ITK, BTK, JAK (JAK1, JAK2 and/or JAK3), IL-9,IL-6, IL-5, IL-13, IL-4, IL-17 (e.g., IL-17A and IL-17F), OX40L, TSLP,IL-25, IL-33, IgE, IL-9 receptor, IL-5 receptor, IL-4 receptor a, IL-13receptor (e.g., IL-13 receptor α1 and/or α2), OX40, TSLP-R, IL-7receptor (e.g., IL7Rα), IL-17 receptor (e.g., IL-17Rβ), ST2 (IL-33receptor), CCR3, CCR4, CRTH2, FcεRI, FcεRII/CD23, Flap, Syk kinase;CCR4, TLR9, CCR3, chemokine receptor antagonist, and GM-CSF. In someembodiments, the method further comprises administering an IL-5antagonist. In some embodiments, the IL-5 antagonist is benralizumab,mepolizumab, and/or reslizumab. In some embodiments, the method furthercomprises administering an IL-13 antagonist. In some embodiments, theIL-13 antagonist is lebrikizumab, dectrekumab, or tralokinumab. In someembodiments, the method further comprises administering an IL-4antagonist (including an IL-4/IL-13 antagonist). In some embodiments,the IL-4 antagonist is dupilumab or QBX-258 (Novartis). In someembodiments, the method further comprises administering a TSLPantagonist. In some embodiments, the TSLP antagonist is AMG-157(MEDI-9929). In some embodiments, the method further comprisesadministering an ST2 antagonist. In some embodiments, the method furthercomprises administering an IL-17 antagonist. In some embodiments, theIL-17 antagonist is secukinumab, ixekizumab or bimekizumab. In someembodiments of any of the methods, the method further comprisesadministering fevipiprant. In some embodiments of any of the methods,the method further comprises administering masitinib. In someembodiments of any of the methods, the method further comprisesadministering a phosphodiesterase (PDE) inhibitor or antagonist, such asa PDE3 and/or PDE4 antagonist. In some embodiments, the PDE antagonistis Theo-24, daliresp, or roflumilast.

In some embodiments of any of the methods, the method further comprisesadministering one or more active ingredients selected from anaminosalicylate; a steroid; a biological; a thiopurine; methotrexate; acalcineurin inhibitor, e.g. cyclosporine or tacrolimus; and anantibiotic. In some embodiments of any of the methods, the methodcomprises administering the further active ingredient in an oral ortopical formulation. Examples of aminosalicylates include4-aminosalicylic acid, sulfasalazine, balsalazide, olsalazine andmesalazine, in forms like Eudragit-S-coated, pH-dependent mesalamine,ethylcellulose-coated mesalamine, and multimatrix-release mesalamine.Examples of a steroid include corticosteroids or glucocorticosteroids.Examples of a corticosteroid include prednisone and hydrocortisone ormethylprednisolone, or a second generation corticosteroid, e.g.budesonide or azathioprine; e.g. in forms like a hydrocortisone enema ora hydrocortisone foam. Examples of biologicais include etanercept; anantibody to tumor necrosis factor alpha, e.g. infliximab, adalimumab orcertolizumab; an antibody to IL-12 and IL-23, e.g. ustekinumab;vedolizumab; etrolizumab, and natalizumab. Examples of thiopurinesinclude azathioprine, 6-mercaptopurine and thioguanine. Examples ofantibiotics include vancornycin, rifaximin, metronidazole, trimethoprim,sulfamethoxazole, diaminodiphenyl sulfone and ciprofloxacin; andantiviral agents like ganciclovir

In some embodiments of any of the methods, the method further comprisesadministering an antifibrotic agent. In some embodiments, theantifibrotic agent inhibits transforming growth factor beta(TGF-β)-stimulated collagen synthesis, decreases the extracellularmatrix, and/or blocks fibroblast proliferation. In some embodiments, theantifibrotic agent is pirfenidone. In some embodiments, the antifibroticagent is PBI-4050. In some embodiments, the antifibrotic agent istipelukast.

In some embodiments of any of the methods, the method further comprisesadministering a tyrosine kinase inhibitor. In some embodiments, thetyrosine kinase inhibitor inhibits a tyrosine kinase that mediateselaboration of one or more fibrogenic growth factors. In someembodiments, the fibrogenic growth factor is platelet-derived growthfactor, vascular endothelial growth factor, and/or fibroblast growthfactor. In some embodiments, the tyrosine kinase inhibitor is imatiniband/or nintedanib. In some embodiments, the tyrosine kinase inhibitor isnintedanib. In some embodiments of any of the methods, the methodfurther comprises administering an antidiarrheal agent. In someembodiments, the antidiarrheal agent is loperamide.

In some embodiments of any of the methods, the method further comprisesadministering an antibody. In some embodiments, the antibody is ananti-interleukin (IL)-13 antibody. In some embodiments, the anti-IL-13antibody is tralokinumab. In some embodiments, the antibody is ananti-IL-4/anti-IL-13 antibody. In some embodiments, theanti-IL-4/anti-IL-13 antibody is SAR 156597. In some embodiments, theantibody is an anti-connective tissue growth factor (CTGF) antibody. Insome embodiments, the anti-CTGF antibody is FG-3019. In someembodiments, the antibody is an anti-lysyl oxidase-like 2 (LOXL2)antibody. In some embodiments, the anti-LOXL2 antibody is simtuzumab. Insome embodiments, the antibody is an anti-av36 integrin receptorantibody. In some embodiments, the anti-av36 integrin receptor antibodyis STX-100. In some embodiments, the antibody is a monoclonal antibody.

In some embodiments of any of the methods, the method further comprisesadministering a lysophosphatidic acid-1 (LPA1) receptor antagonist. Insome embodiments, the LPA1 receptor antagonist is BMS-986020. In someembodiments of any of the methods, the method further comprisesadministering a galectin 3 inhibitor. In some embodiments, the galectin3 inhibitor is TD-139.

In some embodiments of any of the methods, the method further comprisesadministering a palliative therapy. In some embodiments, the palliativetherapy comprises one or more of an antibiotic, an anxiolytic, acorticosteroid, and an opioid. In some embodiments, the antibiotic is abroad-spectrum antibiotic. In some embodiments, the antibiotic ispenicillin, a p-lactamase inhibitor, and/or a cephalosporin. In someembodiments, the antibiotic is piperacillin/tazobactam, cefixime,ceftriaxone and/or cefdinir. In some embodiments, the anxiolytic isalprazolam, buspirone, chlorpromazine, diazepam, midazolam, lorazepam,and/or promethazine. In some embodiments, the corticosteroid is aglucocorticosteroid. In some embodiments, the glucocorticosteroid isprednisone, prednisolone, methylprednisolone, and/or hydrocortisone. Insome embodiments, the opioid is morphine, codeine, dihydrocodeine,and/or diamorphine.

In some embodiments of any of the methods, the method further comprisesadministering an antibiotic. In some embodiments, the antibiotic is amacrolide. In some embodiments, the macrolide is azithromycin, and/orclarithromycin. In some embodiments, the antibiotic is doxycycline. Insome embodiments, the antibiotic is trimethoprim/sulfamethoxazole. Insome embodiments, the antibiotic is a cephalosporin. In someembodiments, the cephalosporin is cefepime, cefixime, cefpodoxime,cefprozil, ceftazidime, and/or cefuroxime. In some embodiments, theantibiotic is penicillin. In some embodiments, the antibiotic isamoxicillin, ampicillin, and/or pivampicillin. In some embodiments, theantibiotic is a penicillin/β-lactamase inhibitor combination. In someembodiments, the penicillin/p-lactamase inhibitor combination isamoxicillin/clavulanate and/or piperacillin/tazobactam. In someembodiments, the antibiotic is a fluoroquinolone. In some embodiments,the fluoroquinolone is ciprofloxacin, gemifloxacin, levofloxacin,moxifloxacin, and/or ofloxacin.

In some embodiments of any of the methods, the method further comprisesadministering a phosphodiesterase inhibitor. In some embodiments, thephosphodiesterase inhibitor is a phosphodiesterase type 5 inhibitor. Insome embodiments, the phosphodiesterase inhibitor is avanafil,benzamidenafil, dasantafil, icariin, lodenafil, mirodenafil, sildenafil,tadalafil, udenafil, and/or vardenafil. In some embodiments, the PDEinhibitor is a PDE-4 inhibitor. In some embodiments, the PDE-4 inhibitoris roflumilast, cilomilast, tetomilast, and/or CHF6001. In someembodiments, the PDE inhibitor is a PDE-3/PDE-4 inhibitor. In someembodiments, the PDE-3/PDE-4 inhibitor is RPL-554.

In some embodiments of any of the methods, the method further comprisesadministering a cytotoxic and/or immunosuppressive agent. In someembodiments, the cytotoxic and/or immunosuppressive agent isazathioprine, colchicine, cyclophosphamide, cyclosporine, methotrexate,penicillamine, and/or thalidomide. In some embodiments of any of themethods, the method further comprises administering an agent thatrestores depleted glutathione levels in the lung. In some embodiments,the agent that restores depleted glutathione levels in the lung isN-acetylcysteine. In some embodiments of any of the methods, the methodfurther comprises administering an anticoagulant. In some embodiments,the anticoagulant is warfarin, heparin, activated protein C, and/ortissue factor pathway inhibitor.

In some embodiments of any of the methods, the method further comprisesadministering an endothelin receptor antagonist. In some embodiments,the method endothelin receptor antagonist is bosentan, macitentan,and/or ambrisentan. In some embodiments of any of the methods, themethod further comprises administering a TNF-α antagonist. In someembodiments, the TNF-α antagonist comprises one or more of etanercept,adalimumab, infliximab, certolizumab, and golimumab. In some embodimentsof any of the methods, the method further comprises administeringinterferon gamma-1b.

In some embodiments of any of the methods, the method further comprisesadministering an interleukin (IL) inhibitor. In some embodiments, the ILinhibitor is an IL-5 inhibitor. In some embodiments, the IL-5 inhibitoris mepolizumab and/or benralizumab. In some embodiments, the ILinhibitor is an IL-17A inhibitor. In some embodiments, the IL-17Ainhibitor is CNTO-6785.

In some embodiments of any of the methods, the method further comprisesadministering a p38 mitogen-activated protein kinase (MAPK) inhibitor.In some embodiments, the p38 MAPK inhibitor is losmapimod and/orAZD-7624. In some embodiments of any of the methods, the method furthercomprises administering a CXCR2 antagonist. In some embodiments, theCXCR2 antagonist is danirixin.

In some embodiments of any of the methods, the method further comprisesvaccination. In some embodiments, the vaccination is vaccination againstpneumococci and/or influenza. In some embodiments, the vaccination isvaccination against Streptococcus pneumoniae and/or influenza. In someembodiments of any of the methods, the method further comprisesadministering an antiviral therapy. In some embodiments, the antiviraltherapy is oseltamivir, peramivir, and/or zanamivir.

In some embodiments of any of the methods, the method further comprisesprevention of gastroesophageal reflux and/or recurrent microaspiration.

In some embodiments of any of the methods, the method further comprisesventilatory support. In some embodiments, the ventilatory support ismechanical ventilation. In some embodiments, the ventilatory support isnoninvasive ventilation. In some embodiments, the ventilatory support issupplemental oxygen. In some embodiments of any of the methods, themethod further comprises pulmonary rehabilitation.

In some embodiments of any of the methods, the method further compriseslung transplantation. In some embodiments, the lung transplantation issingle lung transplantation. In some embodiments, the lungtransplantation is bilateral lung transplantation.

In some embodiments of any of the methods, the method further comprisesa non-pharmacological intervention. In some embodiments, thenon-pharmacological intervention is smoking cessation, a healthy diet,and/or regular exercise. In some embodiments of any of the methods, themethod further comprises administering a pharmacological aid for smokingcessation. In some embodiments, the pharmacological aid for smokingcessation is nicotine replacement therapy, bupropion, and/orvarenicline. In some embodiments, the non-pharmacological interventionis lung therapy. In some embodiments, the lung therapy is pulmonaryrehabilitation and/or supplemental oxygen. In some embodiments, thenon-pharmacological intervention is lung surgery. In some embodiments,the lung surgery is lung volume reduction surgery, single lungtransplantation, bilateral lung transplantation, or bullectomy. In someembodiments, the non-pharmacological intervention is the use of adevice. In some embodiments, the device is a lung volume reduction coil,an exhale airway stent, and/or a nasal ventilatory support system.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of an anti-tryptase antibody, or a pharmaceuticalcomposition thereof, can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent(s). In oneembodiment, administration of an anti-tryptase antibody, or apharmaceutical composition thereof, and administration of an additionaltherapeutic agent occur within about one month; or within about one,two, or three weeks; or within about one, two, three, four, five, or sixdays; or within about 1, 2, 3, 4, 5, 6, 7, 8, or 9 hours; or withinabout 1, 5, 10, 20, 30, 40, or 50 minutes, of each other. Forembodiments involving sequential administration, the anti-tryptaseantibody may be administered prior to or after administration of theadditional therapeutic agent(s).

An anti-tryptase antibody of the invention, or a pharmaceuticalcompositions thereof, (and any additional therapeutic agent) can beadministered by any suitable means, including parenteral,intrapulmonary, and intranasal administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some instances, an anti-tryptaseantibody of the invention may be administered intravitreally,intramuscularly, intravenously, intradermally, percutaneously,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intrathecally, intranasally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularly, mucosally, intrapericardially, intraumbilically,intraocularly, intraorbitally, orally, topically, transdermally,periocularly, conjunctivally, subtenonly, intracamerally, subretinally,retrobulbarly, intracanalicularly, by inhalation, by injection, byimplantation, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, by catheter, by lavage, incremes, or in lipid compositions. In particular instances, the antibody,or a pharmaceutical composition thereof, can be administered bysubcutaneous administration. The compositions utilized in the methodsdescribed herein can also be administered systemically or locally.Dosing can be by any suitable route, for example, by injections, such asintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic. Various dosing schedules includingbut not limited to single or multiple administrations over varioustime-points, bolus administration, and pulse infusion are contemplatedherein.

Antibodies of the invention, or pharmaceutical compositions thereof,would be formulated, dosed, and administered in a fashion consistentwith good medical practice. Factors for consideration in this contextinclude the particular disorder being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody, or pharmaceuticalcomposition thereof, need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg)of antibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 200 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.,every week, every two weeks, every three weeks, or every four weeks(e.g., such that the patient receives from about two to about twenty, ore.g., about six doses of the antibody). For example, a dose may beadministered once per month, (e.g., by subcutaneous injection). Aninitial higher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays. In some instances, a dose of about 50 mg/mL to about 200 mg/mL(e.g., about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL,about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about 170mg/mL, about 180 mg/mL, about 190 mg/mL, or about 200 mg/mL of anantibody may be administered, e.g., by subcutaneous injection. In someinstances, about 150 mg/mL of an antibody may be administered bysubcutaneous injection.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-tryptase antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above (e.g., tryptase-associated disorders) isprovided. The article of manufacture comprises a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, IV solutionbags, etc. The containers may be formed from a variety of materials suchas glass or plastic. The container holds a composition which is byitself or combined with another composition effective for treating,preventing and/or diagnosing the condition and may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Atleast one active agent in the composition is an antibody of theinvention, or a pharmaceutical composition thereof. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises anadditional therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-tryptase antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above. Unless specifically indicated,human tryptase beta 1 was used in the studies.

Example 1: Generation and Humanization of Anti-Tryptase Antibodies

A. Materials and Methods

Residue numbers are according to Kabat et al. Sequences of proteins ofimmunological interest, 5^(th) Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

(i) Recombinant Expression and Purification of Tryptase in Insect Cellsand Mammalian Cells

The sequence encoding mature wild-type human tryptase beta 1(Ile16-Pro246 chymotrypsinogen numbering, SEQ ID NO: 97) was cloned intoa modified pAcGP67A vector behind the polyhedron promoter and the gp67secretion signal sequence. Unless noted otherwise, in this section,tryptase refers to human tryptase beta 1 (also referred to as humantryptase b1). The construct contains an N-terminal His6 tag, anenterokinase cleavage site and, for some constructs, a C-terminal FLAGtag. Site-directed mutagenesis was performed using standard QuikChange™protocols (Stratagene) to generate tryptase mutants. All constructs wereconfirmed by DNA sequencing. Recombinant baculoviruses were generatedusing the BaculoGold™ system (BD Biosciences) in Sf9 cells followingstandard protocols. Trichoplusia ni cells were infected for large-scaleprotein production and harvested 48 h post-infection. The harvestedmedia was supplemented with 1 mM NiCl₂, 5 mM CaCl₂ and 20 mM Tris pH 8,shaken for 30 min, and then centrifuged for 20 min at 8500×g to removethe cells and precipitate from media. The supernatant media was filteredthrough a 0.22 μm polyethersulfone (PES) filter prior to loading onto anickel-nitrilotriacetic acid (Ni-NTA) affinity column. Human tryptasebeta 1 was also expressed by transient transfection in the CHO DP12mammalian cell line. Cell culture media was subjected to the samepurification starting with Ni-NTA affinity purification as describedbelow.

Insect cell media or CHO cell media containing secreted His6-taggedrecombinant tryptase (wild-type or mutant) was loaded onto a 10 mLNi-NTA Superflow column (Qiagen) at a volumetric flow rate of 170 cm/h.The column was washed with 10 CV (column volumes) of wash buffer (20 mMTris pH 8, 10 mM imidazole, 300 mM NaCl) and eluted with 8 CV elutionbuffer (20 mM Tris pH 8, 300 mM imidazole, 300 mM NaCl). Fractionsassayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) containing tryptase were pooled, concentrated, and loadedonto an S200 size exclusion column (GE Healthcare) for furtherpurification using running buffer (10 mM 3-(N-morpholino)propanesulfonicacid (MOPS) pH 6.8, 2 M NaCl) at flow rates recommended by themanufacturer. Fractions containing the His-tagged recombinant tryptase(monomeric) were pooled and concentrated. Recombinant tryptase was thencleaved overnight at room temperature at a concentration of 2 mg/ml inbuffer (10 mM MOPS pH 6.8, 0.2 M NaCl) containing 0.5 mg/ml heparin(Sigma Aldrich) and 0.1 mg/ml enterokinase (New England Biolabs, Inc).This step removes the N-terminal His6-tag and results in tetramerizationand proteolytically active tryptase, which has IVGG as the newly formedN-terminal sequence starting at residue 16 (chymotrypsinogen numbering).Tetrameric tryptase was then subjected to size exclusion chromatographyusing an S200 column (GE Healthcare) in buffer (10 mM MOPS pH 6.8 and 2M NaCl) to purify tetrameric tryptase by removing enterokinase and anyuncleaved recombinant tryptase.

Tryptase mutants Y75C and I99C, as well as the catalytically inactivemutant S195A (chymotrypsinogen numbering, corresponding to Ser224 to Alasubsititution of the full-length tryptase sequence SEQ ID NO: 71) werepurified by Ni-affinity chromatography as described above.Disulfide-linked tryptase dimer mutants were then separated fromnon-disulfide-linked tryptase monomer mutants by S200 size exclusionchromatography. Disulfide-linked dimer mutants were further processed totetramers as described above for wild-type tryptase.

(ii) Anti-Human Tryptase Rabbit and Mouse Monoclonal Antibody Generation

Two rabbits were immunized with CHO-derived human tryptase beta 1(tetramer) with Freund's Complete Adjuvant (CFA). The rabbits wereboosted with the same protein, with Freund's Incomplete Adjuvant (IFA),once every 2 weeks. Following 4 injections, purified serum samples wereevaluated for binding by enzyme-linked immunosorbent assay (ELISA) andinhibition of human tryptase activity using an enzymatic activity assay.B cells from spleen harvested from one of the rabbits, whichdemonstrated inhibition of human tryptase activity, were then fused witha rabbit fusion partner. After 10-14 days, the supernatants wereharvested and screened for protein binding by ELISA.

The ELISA protocol was as follows. 96-well NUNC MAXISORB® ELISA plateswere coated overnight with NeutrAvidin® Biotin Binding Protein (ThermoScientific Catalog No. 31000, stock 10 mg/ml) at 5 μg/ml, 100 μl/well.The wells were washed three times with Wash Buffer (PBS with 0.05%TWEEN®-20) and blotted dry. The wells were then blocked with 200 μl/wellof Blocking Buffer (PBS with 0.5% bovine serum albumin (BSA) and 0.05%TWEEN®-20) and incubated for greater than or equal to 30 min at roomtemperature. Biotinylated human tryptase beta 1 protein (diluted inAssay Buffer; PBS with 0.5% BSA, 0.05% TWEEN®-20, and 0.1 mg/ml heparin)was added to the wells at 1 pg/ml and incubated at room temperature for1 h±10 min. Heparin was included to ensure that tryptase remained as atetramer. The wells were washed three times with wash buffer (200μl/well) and blotted dry. Hybridoma supernatants (samples) or controls(e.g., the positive controls rabbit purified polyclonal antibody YZ4209(stock 0.25 mg/ml) were added to the wells (diluted in Assay Buffer, 100μl/well) and incubated at room temperature for 1 h. Next, the wells werewashed three times with wash buffer (200 μl/well) and blotted dry.Horseradish peroxidase (HRP) conjugate (goat anti-rabbit IgG (H+L) HRP;Thermo Scientific Catalog No. 31460) was added (diluted 1:10,000 inAssay Buffer, 100 μl/well) and incubated at room temperature for 30 min.The wells were again washed three times with wash buffer (200 μl/well)and blotted dry. The substrate (BioFX TMB Substrate, Product No.:TMBW-1000-01) was added (100 μl/well) and incubated for 5 min at roomtemperature. The reaction was stopped by addition of 100 μl/well ofBioFX stop solution (Product No.: BSTP-0100-01), and the plates wereread at A₆₅₀.

All ELISA-positive clones were purified by affinity chromatography usingstandard methods (MabSelect SuRe™; GE Healthcare) and then screened forinhibition of human tryptase activity in a recombinant tryptase activityassay. Briefly, antibodies were diluted from 0.007 to 100,000 ng/mL(0.046 pM to 667 nM) in PBS, pH 7.4. Recombinant human tryptase beta 1was diluted to 3 nM in TNH Buffer (200 mM Tris, 150 mM NaCl, 0.1 mg/mLheparin, 0.01% TRITON™ X-100, pH 8.0) and combined 1:1 withanti-tryptase antibodies in black 384-well plates (ViewPlate-384 F,Black, Clear-Bottom, Perkin Elmer, Catalog No. 6007470). Plates wereincubated for 1 h at ambient temperature with gentle agitation.Colorimetic substrate S-2288 (Chromogenix, Part No. 82-0852-39) wasdiluted to 900 pM in TNH Buffer and was added to the plate. Finalin-well concentrations were 300 pM S-2288, 1 nM recombinant humantryptase beta 1, and 0.015 pM to 222 nM anti-tryptase antibodies. Plateswere incubated for 40 min at ambient temperature with gentle agitationand then were read at A4₀. The half-maximal inhibitory concentration(IC50) of the anti-tryptase antibodies were determined from afour-parameter fit of their respective curves. Clones demonstrating thedesired inhibition were then subcloned by limiting dilution (singlecell/well), retested as described above and further characterized.

Mouse hybridomas were generated and screened and positive clones wereanalyzed in a similar manner. Three A/J mice (Harlan, Indianapolis,Ind.) were immunized with purified tryptase protein as the antigen (EPC,Inc. # TR913). Enzymatically active tryptase as administed at 20pg/mouse per immunization subcutaneously and intraperitoneally aftermixing and emulsifying 1:1 with Complete Freund's Adjuvant (firstimmunization) or Incomplete Freund's Adjuvant (second, third, and fourthimmunization). The boost (fifth immunization) had no adjuvant. After thefourth immunization, sera from immunized mice were tested by ELISA andthe mouse with serum titer higher than OD₄₅₀>2.16 at the dilution of1:1.28×10⁴ was selected for hybridoma fusion. 107×10⁶ isolated spleencells were mixed with 100×10⁶ Sp2/0 myeloma cells (ATCC) for fusion inthe presence of polyethylene glycol (Sigma-Aldrich, Cat. No. P7777-5G).Twenty-four clones were screened for binding to tryptase and assayed forinhibition of tryptase enzymatic activities. Clone T31a (also referredto herein as “31A” and “mu.31A”) was selected for humanization.

(iii) Molecular Cloning and Reformatting of Rabbit Anti-TryptaseHybridoma Clones

Total RNA was extracted from hybridoma cells producing the rabbitanti-tryptase monoclonal antibodies (RNeasy® Mini Kit, Qiagen). UsingSMARTer® RACE cDNA Amplification Kit (Clontech), the RNA was firstreverse transcribed, then subjected to first strand cDNA synthesis and5′ RACE PCR amplification of variable light (VL) and variable heavy (VH)domains with the following primers:

Light chain (LC) forward primer:

Universal Primer Mix (SMARTer® RACE cDNA Amplification Kit, Clontech,catalog #634858)

Heavy chain (HC) forward primer:

Universal Primer Mix (SMARTer® RACE cDNA Amplification Kit, Clontech,catalog #634858)

LC reverse primer: (SEQ ID NO: 74) 5′-GATGGTGACTGTTCCAGTTGC-3′HC reverse primer: (SEQ ID NO: 75) 5′-CATTGGTGAGGGTGCCCGAGTTC-3′The LC and HC reverse primers were designed to anneal to a region in theconstant light (CL) and constant heavy domain 1 (CH1).

Amplified PCR products were directly sequenced. The identified VL DNAsequence was then subcloned into pRK mammalian cell expression vectorcontaining the human kappa constant domain. The VH DNA sequence wasinserted into pRK vectors encoding the full-length human γ1 constantdomain.

(iv) Humanization of the Rabbit Anti-Tryptase Monoclonal Antibody E104

The VL (SEQ ID NO: 53) and VH (SEQ ID NO: 52) domains from the rabbitanti-tryptase monoclonal antibody E104 were aligned with the human VLkappa I (VL_(KI)) and human VH subgroup IV (VH_(IV)) consensus sequences(SEQ ID NOs: 92 and 93, respectively). See, e.g., Dennis, Ch. 2 CDRRepair: A Novel Approach to Antibody Humanization in Current Trends inMonoclonal Antibody Development and Manufacturing, Eds. Shire et al.Springer, New York, N.Y. The hypervariable regions (HVR) were engineeredinto the consensus human VL_(KI) and Vh_(IV) acceptor frameworks togenerate CDR-graft variants. From the E104 VL domain, positions 24-34(L1), 50-56 (L2) and 89-97 (L3) were grafted into VL_(KI). From the VHdomain, positions 26-35b (H1), 50-65 (H2) and 93-102 (H3) were graftedinto VH_(IV). To evaluate framework vernier positions that might beimportant, selected vernier positions were mutated back to the rabbitsequences. The vernier positions that were mutated back to the rabbitsequences included positions 2, 4, 43, 68, and 87 in VL and 37, 67, 71,78, and 91 in VH.

The VL and VH domains from the rabbit anti-tryptase monoclonal antibodyE104 were also aligned with the human VL kappa I (VL_(KI)) and human VHsubgroup III (VH_(III)) consensus sequences (SEQ ID NOs: 92 and 94,respectively). See Dennis, supra. The hypervariable regions (HVR) wereengineered into the consensus human VL_(KI) and VH_(III) acceptorframeworks to generate CDR-graft variants. From the rab.E104 VL domain,positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) were grafted intoVL_(KI). From the VH domain, positions 26-35 (H1), 50-65 (H2), and93-102 (H3) were grafted into VH_(III).

In total, two different versions of humanized VL sequences and sixdifferent versions of humanized VH sequences were synthesized andsubsequently subcloned into pRK mammalian expression vectors. Bycombining the different versions of the LC and HC, a total of twelvedifferent humanized E104 variants (v1 to v12) were generated (Table 3).All the humanized anti-tryptase variants were expressed as IgG1 or IgG4antibodies in mammalian cells. Antibodies were purified by affinitychromatography using standard methods (MabSelect SuRe™; GE Healthcare).All of the IgG4 antibodies used in the Examples section included anS228P mutation (EU numbering) in the heavy chain constant region;however, the invention described herein is not limited to the IgG4variant with the S228P mutation.

A DNA sequence encoding the VH domain of huE104.v2 is shown in SEQ IDNO: 109. A DNA sequence encoding the VL domain of huE104.v2 is shown inSEQ ID NO: 110. A DNA sequence encoding the heavy chain (IgG1) is shownin SEQ ID NO: 111. A DNA sequence encoding the heavy chain (IgG4.S228P)is shown in SEQ ID NO: 113. A DNA sequence encoding the light chain(IgG1 and IgG4) is shown in SEQ ID NO: 112. The amino acid sequence ofthe heavy chain (HC) of huE104.v2 IgG1 is shown in SEQ ID NO: 80. Theamino acid sequence of the light chain (LC) of huE104.v2 (IgG1 or IgG4)is shown in SEQ ID NO: 81. The amino acid sequence of the HC ofhuE104.v2 IgG4 S228P is shown in SEQ ID NO: 82.

(v) Humanization of the Mouse Anti-Tryptase Monoclonal Antibody 31A

The VL (SEQ ID NO: 20) and VH (SEQ ID NO: 19) domains from the mouseanti-tryptase monoclonal antibody 31A (“mu.31A”) were aligned with thehuman VL kappa I (VL_(KI)) and human VH subgroup III (VH_(III))consensus sequences (SEQ ID NOs: 92 and 93, respectively). Thehypervariable regions (HVR) were engineered into the consensus humanVL_(KI) and VH_(III) acceptor frameworks to generate CDR-graft variants.From the mu.31A VL domain, positions 24-34 (L1), 50-56 (L2), and 89-97(L3) were grafted into VL_(KI). From the mu.31A VH domain, positions26-35 (H1), 50-65 (H2), and 93-102 (H3) were grafted into VH_(III). Toevaluate the importance of the framework vernier positions, selectedvernier positions were mutated back to the mouse sequences. The vernierpositions that were mutated back to the mouse sequences includedpositions 4, 43, 46, 47, and 71 in VL and position 49 in VH.

In summary, three humanized LC and five humanized HC were synthesizedand subsequently subcloned into pRK mammalian expression vectors. Bycombining the different versions of the LC and HC, a total of fifteendifferent humanized variants (v1 to v15) of 31A (“hu31A”) were generated(Table 4).

All the humanized anti-tryptase variants were expressed as IgG1 or IgG4antibodies in mammalian cells. Antibodies were purified by affinitychromatography using standard methods (MabSelect SuRe™; GE Healthcare,Piscataway, N.J., USA).

A DNA sequence encoding the VH domain of hu31A.v11 is shown in SEQ IDNO: 104. A DNA sequence encoding the VL domain of hu31A.v11 is shown inSEQ ID NO: 105. A DNA sequence encoding the heavy chain (IgG1) is shownin SEQ ID NO: 106. A DNA sequence encoding the heavy chain (IgG4.S228P)is shown in SEQ ID NO: 108. A DNA sequence encoding the light chain(IgG1 and IgG4) is shown in SEQ ID NO: 107. The amino acid sequence ofthe HC of hu31A.v11 IgG1 is shown in SEQ ID NO: 76. The amino acidsequence of the LC of hu31A.v11 IgG1 is shown in SEQ ID NO: 77. Theamino acid sequence of the HC of hu31A.v11 IgG4 S228P is shown in SEQ IDNO: 78. The amino acid sequence of the LC of hu31A.v11 IgG4 S228P isshown in SEQ ID NO: 79.

(vi) Cloning, Expression, and Purification of Fab Fragments

Fabs were cloned and expressed in E. coli as previously described (seeSimmons et al. J. Immunol. Methods 263:133-147, 2002; Lombana et al.Sci. Rep. 5:17488, 2015). E. coli cell paste containing the expressedFab was harvested from fermentations expressing Fabs and dissolved intoPBS buffer containing 25 mM EDTA and 1 mM phenylmethylsulfonyl fluoride(PMSF). The mixture was homogenized and then passed twice through amicrofluidizer. The suspension was then centrifuged at 21,500×g for 60min. The supernatant was then loaded onto a Protein G columnequilibrated with PBS at 5 ml/min. The column was washed with PBS bufferto baseline and proteins were then eluted with 0.6% acetic acid.Fractions containing Fabs as assayed by SDS-PAGE were pooled and thenloaded onto a 50 mL SP SEPHAROSE® column equilibrated in 20 mM MES (pH5.5). The column was washed with 20 mM MES buffer (pH 5.5) for 2 columnvolumes and then eluted with a linear gradient to 0.5M NaCl in 20 mM MESbuffer (pH 5.5). For final purification, Fab-containing fractions fromthe ion exchange chromatography were concentrated and run on a S75 sizeexclusion column in PBS buffer. The same protocol was used for all Fabsused in the experiments.

(vii) BIAcore® Surface Plasmon Resonance (SPR) Analysis of HumanizedAnti-Tryptase Variants

In this experiment, all the humanized variants were expressed as IgG bytransient transfection of 293 cells. IgG was purified with protein Gaffinity chromatography. The affinity of each variant for recombinantHis-tagged human tryptase beta 1 monomer (SEQ ID NO: 128) was determinedby SPR analysis using a BIAcore® T200. BIAcore® Series S CM5 sensorchips were immobilized with monoclonal mouse anti-human IgG (Fc)antibody (Human antibody capture kit from GE Healthcare) andanti-tryptase variants were subsequently captured on each flow cell.Serial 3-fold dilutions of the human tryptase beta 1 monomer wereinjected at a flow rate of 30 μl/min. Each sample was analyzed with 3min association and 10 min dissociation. After each injection, the chipwas regenerated using 3 M MgCl₂. Binding response was corrected bysubtracting the response units (RU) from a flow cell capturing anirrelevant IgG at similar density. A 1:1 Languir model of simultaneousfitting of k_(on) and k_(off) was used for kinetics analysis.

(viii) Analysis of Inhibitory Activity of Humanized Anti-TryptaseVariants

a. Tryptase Enzymatic Assay

The inhibition of human tryptase activity by humanized anti-tryptaseantibodies was measured using a recombinant tryptase activity assay.hu31a.v11 IgG4 and hu31a.v11 IgG1 were diluted from 0.05 to 100 μg/ml(0.30 to 667 nM) in PBS, pH 7.4, and huE104.v2 IgG4 and huE104.v2 IgG1were diluted from 0.02 to 50 μg/ml (0.15 to 333 nM) in PBS pH 7.4.Recombinant human tryptase beta 1 tetramer active enzyme was diluted to0.75 nM in TNH Buffer (200 mM Tris, 150 mM NaCl, 0.1 mg/mL heparin,0.01% TRITON™ X-100, pH 8.0), and combined 1:1 with anti-tryptaseantibodies in black 384-well plates (ViewPlate-384 F, Black,Clear-Bottom, Perkin Elmer, Catalog No. 6007470). Plates were incubatedfor 1 h at ambient temperature with gentle agitation. Colorimetricsubstrate S-2288 (Chromogenix, Part No. 82-0852-39) was diluted to 1200μM in TNH Buffer and was added to the plate. Final in-wellconcentrations were 400 μM S-2288, 0.25 nM recombinant human tryptasebeta 1 tetramer, 66 μg/mL heparin, and 0.10 to 222 nM anti-tryptaseantibodies for hu31A.v11 and 0.05 to 111 nM for huE104.v2. Plates wereincubated for 40 min at ambient temperature with gentle agitation andthen were read at A₄₀₅. The IC50 of the anti-tryptase antibodies weredetermined from a four-parameter fit of their respective curves.

b. Bronchial Smooth Muscle Cell Proliferation Assay and Collagen-BasedContraction Assay

Human bronchial smooth muscle cells (BSMCs; Catalog No. CC-2576, LonzaMinneapolis, Minn.) were cultured in a humidified incubator at 37° C.with 5% CO₂ in complete culture media SmGM-2 (Catalog No. CC-3182,Lonza). The assay media was SmGm-2 culture media without human serum orsupplements added (Catalog No. CC-3181, Lonza). Human wild-type tetramertryptase or human S195A catalytically-inactive tryptase enzyme(chymotrypsinogen numbering) were used at the specified concentrations.Anti-human tryptase antibodies hu31A.v11 IgG4 and E104.v2 IgG4 were usedat the specified concentrations.

For the proliferation assay, 1 day prior to performing the assay, BSMCswere plated at 2×10⁵ cells/ml in a 96 well tissue culture plate (CatalogNo. 353072, Falcon BD) in complete culture media. After 24 h, theculture media was replaced with assay media and cells were incubated foran additional 24 h. Anti-human tryptase antibodies were serially diluted3.3-fold in assay media in a 96 well tissue culture plate (Catalog No.353072, Falcon BD). 100 μL of diluted hu31A.v11 IgG4 was transferred toa 96 well plate containing 100 μL of 200 nM human tryptase. Activetryptase and anti-human tryptase antibodies were incubated for 30 min atroom temperature. At this time, assay media was removed from the platedcells and replaced with 100 μL of the diluted antibodies plus tryptase.The final concentration of antibodies ranged from 2.0 μM to 4 pM. Thefinal concentration of tryptase was 100 nM (without heparin). The finalsalt concentration was about 130 mM. Wells with tryptase alone wereincluded as stimulation controls. Wells with assay media alone wereincluded as unstimulated controls. Plates were incubated for 24 h at 37°C. before the addition of 1 μCi of H³-thymidine per well. After anadditional 6 h of incubation, proliferation was measured by H³-thymidineincorporation. Cell-associated radioactivity was quantified byscintillation counting. Results were expressed as the mean of triplicatesamples. Graphs were generated and statistical analysis was performedusing KaleidaGraph (Synergy Software).

For the collagen-based contraction assay, 1 day prior to performing theassay, BSMCs were plated in collagen at 9×10⁶ cells/mL in a 24 wellplate (Catalog No. 353047, Falcon BD) following the manufacturer'sguidelines (Catalog No. CBA-201, Cell BioLabs Inc.). After a 1 hincubation at 37° C., cells were overlayed with 1 mL of assay media.After a 24 h incubation at 37° C., media was replaced with 250 μL offresh assay media. Tryptase was diluted in 250 μL assay media to 660 nMin the presence or absence of 4 pM of anti-human tryptase antibodies andincubated for 30 min at 37° C. prior to adding to the specified wellscontaining the cell:collagen matrix. The final concentrations were 330nM tryptase and 2 μM antibody (without heparin). The final saltconcentration was about 130 mM. Cell contraction was initiated by therelease of the cell:collagen matrix from the plate wall using a sterilepipette tip. Assay media alone was used as an unstimulated control. Atthe start of cell contraction (t=0), cell:collagen matrices werevisualized, imaged, and recorded using a ProteinSimple Alphalmager®.Cells were incubated at 37° for an additional 3 h and cell:collagenmatrices were reimaged and recorded (t=3). Data was analyzed using NIHImage J software. Data were represented as the percent change incell:collagen matrix diameter from the start of contraction (t=0) untilthe 3 h time point (t=3). Results were expressed as the mean oftriplicate samples.

c. Mast Cell Histamine Release Assay

The human mast cell line LAD2 was used. LAD2 cells were cultured in ahumidified incubator at 37° C. with 5% CO₂ in serum-free growth medium,StemPro®-34, containing StemPro®-34 nutrient supplement (Catalog No.10640-019, Gibco/Life Technologies), 1× peniciin-streptomycin-glutarnine(Catalog No. 10378-016, Gibco/Life Technologies) and 100 ng/mLrecombinant human stem cell factor (SCF) (Catalog No. 573908,BioLegend). See Kirshenbaum et al. Leukemia Research 27:677-682, 2003.Human tryptase wild-type or human tryptase S195A mutant were used at thespecified concentrations. Anti-NP human IgE (JW8.5.13; see Jackman etal. J. Biol Chem. 285(27):20850-20859, 2010) was obtained from SerotecInc. NP-BSA (Catalog No. N5050H-10, Biosearch Technologies, Petaluma,Calif.) was used at the specified concentration for triggering histaminerelease. Anti-human tryptase antibodies hu31A.v11 IgG4 and E104.v2 IgG4were used at the specified concentrations. The small molecule inhibitorG02849855 was used at the specified concentration. Cell stimulationswere performed using Tyrode's Salt (Catalog No. T-2397, Sigma).Histamine was measured using a histamine ELISA kit (GenWay. Catalog No.40-371-25010).

For the human IgE triggered histamine release assay, LAD2 cells wereplated at 4×10⁶ cells/4 ml in 2 wells of a 6 well dish. Anti-NP IgE (100ng/ml) was added to one well and the cells were incubated overnight at37° C. to prime the cells. In the other well, cells were cultured inmedia only without the addition of anti-NP IgE to serve as a negativecontrol. After the overnight incubation, cells were washed 3 times withcell culture media to remove the unbound IgE. Cells were resuspended in4 mL Tyrode's salts (cell density 1×10⁶ cells/ml) and aliquoted intoEppendorf® tubes (300,000 cells/tube). Samples were incubated with 100μg/mL hu31A.v11 IgG4 or 10 μM G02849855, mixed thoroughly, and incubatedat room temperature for 1 h. Following this incubation, NP-BSA was addedto the samples at a final concentration of 0.1 μg/mL to trigger celldegranulation. Samples were mixed thoroughly and incubated at 37° C. ina CO₂ incubator for 1 h. Cells were then centrifuged at 3000 rpm for 5min at room temperature, and the supernatant was collected for histaminemeasurement. Histamine in the degranulation supernatant was quantitatedusing a histamine ELISA kit. Data were represented as the mean ofduplicate samples.

For the human tryptase triggered histamine release assay, LAD2 cellswere resuspended in Tyrode's salts at a concentration of 10⁶ cells/mLand aliquoted into Eppendorf® tubes (300,000 cells/tube). To promotecell degranulation, cells were treated with 3 μg/mL tryptase (wild-typeor S195A mutant) or 3 μg/mL tryptase which had been pre-incubated with100 μg/mL hu31A.v11 for 45 min at room temperature. PBS was used as a nostimulation control. Samples were mixed thoroughly and incubated at 37°C. in a CO₂ incubator for 1 h. The final salt concentration was about140 mM. Cells were then centrifuged at 3000 rpm for 5 min at roomtemperature, and the supernatant was collected for histaminemeasurement. Histamine in the degranulation supernatant was quantitatedusing a histamine ELISA kit. Data were represented as the mean ofduplicate samples.

(ix) Purification of Recombinant Tryptase Monomers and Tetramers

Human tryptase without the endogenous signal peptide (amino acidresidues 1-15 of SEQ ID NO:71) and pro-peptide (amino acid residues16-30 of SEQ ID NO:71) was expressed in mammalian CHO cells as aHis-tagged recombinant protein with an engineered enterokinase cleavagesite and underwent 5x ultrafiltration followed by a 5x dilution intoPBS. Media was then loaded over a Ni-NTA column and eluted with 250 mMimidazole. Eluate was dialyzed into 10 mM MOPS, 0.2M NaCl, pH 6.8 bufferyielding monomeric tryptase. Uncleaved His6-tagged tryptase monomersremain monomeric and do not form tetramers.

To generate tetrameric tryptase, monomeric tryptase containing the Histag was digested using 0.1 mg/ml of enterokinase enzyme and stabilizedwith dextran sulfate-10 sodium salt at 0.5 mg/ml for 16-20 h at roomtemperature. Protein was passed over a SUPERDEX™ 200 column into thefinal buffer of 10 mM MOPS, 2M NaCl, pH 6.8 to separate tetramers frommonomers and from any residual contaminating protease. Exemplarypurified tetramer is shown in FIG. 3A, peak 1. Pooled tetramerictryptase was used for immunization and activity assays.

B. Results

(i) Hybridoma Cloning

Molecular cloning of five rabbit anti-tryptase antibodies fromELISA-positive hybridoma clones revealed four unique anti-tryptaseclones. Of these, clone E104 showed the most robust inhibition activityin the enzymatic assay, and was selected for further engineering. Inparallel, a murine anti-tryptase monoclonal antibody clone 31A whichshowed inhibition activity in the enzymatic assay was also selected forfurther engineering.

(ii) Generation of Chimeric E104 (chE104) Variants

The light chain of the rabbit anti-tryptase monoclonal antibody E104 isa rabbit kappa light chain, which contains a disulfide bridge betweenCys80 in FR3 of the variable domain (VL) and Cys170 in the constantdomain (CL). To evaluate the importance of Cys80 in the VL, a chimericE104 variant with the cysteine at position 80 mutated to alanine(Cys80Ala) was generated. As shown in Table 2, no difference between thetwo variants in terms of yield or aggregation, as evidenced by the high% monomer in both the Cys80 and the Cys80Ala chimeric variantsdetermined by size exclusion chromatography. Thus, it was determinedthat the Cys80 residue was not critical and in the humanized version,Cys80 was changed to a proline residue as in the VH4 graft. In addition,chimeric E104 antibodies with the Cys80Ala mutation in the variabledomain FR3, either fused to the human IgG1 or IgG4 constant domains,showed similar inhibitory activities as the rabbit monoclonal antibodywith cysteine at position 80 (data not shown).

TABLE 2 Effect of Cys80 Residue of Anti-Tryptase Antibody E104 on Yieldand Aggregation Light chain Anti-tryptase variants (position Yield %clone 80 residue) (mg) monomer E104 chE104.C 3.01 95.45 chE104.A 3.1495.2

(iii) Humanization of the Rabbit Anti-Tryptase Monoclonal Antibody CloneE104 and the Mouse Anti-Tryptase Monoclonal Antibody Clone 31A

All of the humanized variants were expressed as IgG and their bindingaffinities were evaluated in a BIAcore® SPR assay. In general, all ofthe humanized E104 variants (huE104) showed similar binding affinitiestowards the human tryptase beta 1 monomer (Table 3). Table 3 listshuE104 variants as well as the binding affinity (in terms of K_(D)) asdetermined by BIAcore® SPR analysis. Table 3 also provides the SEQ IDNOs of the VH and VL domains for each variant. Each clone in Table 3 wastested in IgG1 format. Consistently, very similar IC50 values wereobserved for these antibodies in the enzymatic activity assay describedabove, except that the huE104.v1 and huE104.v5 clones, and to a lessextent, huE104.v11 and huE104.v12, showed some “hook effect” (in whichan increase of enzymatic activity, i.e., a decrease in antibodyinhibitory activity, was observed at high antibody concentrations) inthe enzymatic assay. Modifications in the heavy chain FR3 region fromV71 of the humanized antibody to the original Arg residue of the rabbitmonoclonal antibody (V71R) and F78 of the humanized antibody to theoriginal Val residue of the rabbit monoclonal antibody (F78V) eliminatedthe hook effect seen in E104.v1, v5, v11 and v12. The E104.v9 chimericclone contains R71 and V78 as in the parent rabbit monoclonal Ab E104.See Table 3. As shown in FIG. 12B, the arginine (R) residue at position71 and valine (V) residue at position 78 (both Kabat numbering) may playan important role in conformation of HVR-H2, and thus the binding of theantibody to tryptase. As a result, the V71R and F78V modifications inhuE104.v2 may have affected the conformation of the tryptase 80 loopthat is important in the association of two protomers that form thesmall interface. See Example 3 below. Thus, hu104.v2, which has the V71Rand F78V reversions, has improved binding and inhibitory activity ascompared to v1, which has V at position 71 and F at position 78. Allvariants except v1, v5, v11, and v12 showed complete inhibition oftryptase activity as measured by the enzymatic assay described above.The humanized clone huE104.v2 was selected for further evaluation. Theamino acid sequences of the heavy and light chain variable domains ofhuE104.v2 are shown in FIG. 1.

TABLE 3 Binding affinities of humanized E104 (huE104) variants (bindingaffinity to human tryptase b1 monomer in parentheses) Light Chain K1graft +A2 +L4 K1 graft +P43 +E68 +F87 Chimeric LC (SEQ ID NO: 37) (SEQID NO: 58) (SEQ ID NO: 59) Heavy Chain VH4 graft v1 (0.43 nM) v5 (0.18nM) — (SEQ ID NO: 47) VH4 graft v2 (0.18 nM) v6 (0.14 nM) v10 (0.26 nM)+R71 +V78 (SEQ ID NO: 36) VH4 graft v3 (0.21 nM) v7 (0.25 nM) — +V37+S67 +R71 +V78 +F91 (SEQ ID NO: 48) VH3 graft v4 (0.44 nM) v8 (0.24 nM)— +I48 +S67 +T73 +V78 (SEQ ID NO: 51) Chimeric HC v9 (0.24 nM) — — (SEQID NO: 52) VH4 graft v11 (0.32 nM) — — +V78 (SEQ ID NO: 49) VH4 graftv12 (0.12 nM) — — +R71 (SEQ ID NO: 50)

Table 4 lists hu31A variants as well as the binding affinity (in termsof K_(D), nanomolar) as measured by BIAcore® SPR analysis. Table 4 alsoshows the SEQ ID NOs of the V_(H) and V_(L) for each antibody. Allvariants showed complete inhibition of tryptase activity as measured bythe enzymatic assay (data not shown). Each clone in Table 4 was testedin IgG1 format. The clone hu3 Å.v11 showed the best affinity and bestinhibitory activity in anenzymatic assay and was therefore selected forfurther evaluation. The amino acid sequences of the heavy and lightchain variable domains of hu31AMv11 are shown in FIG. 1.

TABLE 4 Binding affinities of humanized 31A (hu31A) variants (bindingaffinity to human tryptase b1 monomer in parentheses) Light Chain K1graft K1 graft +L4 +S43 +P46 K1 graft +S43 +P46 +W47 +W47 +Y71 (SEQ IDNO: 102) (SEQ ID NO: 10) (SEQ ID NO: 103) Heavy VH3 graft v1 (4.04 nM)v2 (0.74 nM) v3 (0.79 nM) Chain (SEQ ID NO: 98) VH3 graft v4 (11.8 nM)v5 (1.68 nM) v6 (1.65 nM) +A49 (SEQ ID NO: 99) VH3 graft v7 (2.06 nM) v8(0.94 nM) v9 (0.94 nM) +S31 +F32 +H35 +A49 (SEQ ID NO: 100) VH3 graftv10 (1.19 nM) v11 (0.40 nM) v12 (0.43 nM) +A49 +T93 +N96 +Y97 +D98 (SEQID NO: 9) VH3 graft v13 (9.65 nM) v14 (1.34 nM) v15 (1.32 nM) +A49 +T93(SEQ ID NO: 101)

The inhibitory activity of humanized anti-tryptase antibodies was alsodetermined using the recombinant tryptase enzymatic activity assaydescribed above. Both h31A.v11 and huE104.v2, IgG1 and IgG4, completelyinhibited tryptase activity in the enzymatic assay (see FIG. 2A). TheIC50 values are shown below (Table 5).

TABLE 5 Inhibitory activity (IC50) of humanized anti-tryptase antibodiesAntibody IgG1 IgG4 hu31A.v11 1.82 nM ± 0.09  3.9 nM ± 0.65 huE104.v20.91 nM ± 0.35 0.59 nM ± 0.11

Both hu31Av11 and huE104.v2 bind and inhibit human tryptase beta 2andbeta 3, in addition to tryptase beta 1. Representative data are shownbelow in Table 6 based on protocols described above for affinityanalysis and enzymatic assay using human tryptase beta 1 as the target.Both hu31A.v11 and huE104.v2 also bind and inhibit cyno tryptase D1.

TABLE 6 Affinity and IC50 of humanized anti-tryptase antibodies AntibodyHuman Tryptase K_(D) (nM) IC50 (nM) hu31A.v11 IgG4 Tryptase beta 1 0.273.16 Tryptase beta 2 0.12 1.46 Tryptase beta 3 0.10 2.47 huE104.v2 IgG4Tryptase beta 1 0.3 0.42 Tryptase beta 2 0.18 0.95 Tryptase beta 3 0.132.80

The inhibitory activity of hu31A.v1 IgG4 or huE104.v2 IgG4 was furtherassessed in several ex vivo models of human primary airway smooth musclecell (SMC) function. Addition of tryptase beta 1 to culture mediumresults in an increase in proliferation of human primary airway SMCs(FIG. 2B), as well as contraction of the cells (FIG. 2C). Addition ofhu31A.v1 or huE104.v2 resulted in a dose-dependent reduction inproliferation, and at 300 μg/ml inhibited proliferation to baselinelevels (FIG. 2B). Similarly, addition of hu31A.v11 or huE104.v2 reducedcontraction to baseline levels (FIG. 2C). These data demonstrate thatthe anti-tryptase antibodies hu31A.v1 and huE104.v2 inhibited tryptasefunction.

Addition of tryptase or IgE to mast cells results in degranulation andrelease of histamine (FIGS. 2D-2E). The ability of hu31A.v1 to inhibithistamine release was assessed. Tryptase beta 1 with the S195Asubstitution is catalytically inactive. Addition of hu31A.v11 blockedthe ability of tryptase to promote histimine release (FIGS. 2D-2E). Theextent of inhibition (30-50%) was similar to the small moleculeinhibitor of tryptase beta 1 activity, G02849855 (FIG. 2E). Theseresults further show that the anti-tryptase antibody hu31A.v1 inhibitstryptase function.

Example 2: hu31A.v1 and huE104.v2 IgG Dissociate Human Tryptase BetaTetramer

The crystal structure of active tetrameric tryptase shows that thecatalytic site of each protomer (represented by S195, H57, and D102 ofeach protomer, chymotrypsinogen numbering) is located inside the pore ofthe tetramer, and that access to the active sites is limited such thatonly peptide substrates and smaller molecules can gain access (Pereiraet al. Nature 392:306-11, 1999). To date, no mammalian serine proteaseinhibitors are known to inhibit the proteolytic activity of tryptase.Human serine protease inhibitors of the Kunitz-domain type architectureare too large to access the active sites. There are no known naturalinhibitors of tryptase in humans. The only known natural macromolecularinhibitors of tryptase to date are leech-derived tryptase inhibitor(LDTI) (Sommerhoff et al. Biol. Chem. 373:685-94, 1997) and tick-derivedprotease inhibitor (TdPI) (Paesen et al. J. Mol. Biol. 368:1172-86,2007). LDTI (4.7 kDa) and TdPI (11.1 kDa) have the ability to block twoor three of the four active sites of the tryptase tetramer,respectively. LDTI and TdPI do not dissociate the tryptase tetramer.Both the IgG and Fab of hu31A.v11 are much larger than LDTI or TdPI, andyet they completely inhibit tryptase.

We determined the stoichiometry of Fab hu31A.v11 binding to tetramerictryptase in solution. Active tetrameric tryptase beta 1 was mixed with a2-fold molar excess of Fab hu31A.v11 to each protomer and complexformation was allowed to reach equilibrium. Because the tetramer isassembled non-covalently, the dissociating effect of each antibody onthe tetrameric structure was analyzed by size exclusion chromatography(SEC) and the retention times/volumes and the molecular weights of theindividual protein peaks were determined. The fractions containingprotein were characterized by SDS-PAGE to determine the proteincomponents (FIG. 3A). The retention time of tetrameric tryptase alonewas significantly shorter (t_(r)=26 min, run 1, peak 1) than when incomplex with Fab hu31A.v1 (t_(r)=28.1 min, run 3, peak 3). An additionalSEC analysis in combination with multi-angle light scattering (MALS) wasperformed to determine the molecular weight of the eluted proteincomplexes. Tetrameric tryptase had a molecular weight of 120 kDa±0.2%according to MALS, which is consistent with the theoretical molecularweight of 109.537 kDa based on amino acid sequence (excludingglycosylation). The protein peak containing tryptase in complex with Fabhu31A.V11 was measured to be 67.7 kDa±3% by MALS (peak 3), whichreflects a complex comprising 1 tryptase monomer bound to 1 Fab. Thisindicates that the tryptase tetramer dissociates into monomers uponbinding to Fab hu31A.v11, which is further corroborated by the crystalstructure (see Example 3) and the inhibitory activity of the hu31A.v11Fab.

When the same tetrameric tryptase/Fab hu31A.v11 complex was analyzed bySEC in enzyme assay buffer that contained a high concentration ofheparin, e.g., 100 μg/ml heparin (run 2), the retention time of thetryptase-Fab complex was only slightly decreased from 28.1 min (run 3,peak 3) to 27.6 min (run 2, peak 2), likely due to heparin binding tothe protein complex of tryptase monomer and Fab. Heparin from porcineintestinal mucosa is a mixture of polyanion chains having molecularweights ranging from 6 to 30 kDa, with most chains in the range of 17 to19 kDa. This result indicates that the stabilizing effect of heparin ontetrameric tryptase is also neutralized or disrupted by Fab hu31A.v1;even a high concentration 100 μg/ml of heparin cannot prevent Fabhu31A.v11 from completely dissociating the tetramer. See FIG. 3A.

The binding stoichiometry of the huE104.v1 and huE104.v2 Fabs totetrameric tryptase in solution was also determined. Active tetramerictryptase was mixed with a 2-fold molar excess of Fab to each protomerand complex formation was allowed to reach equilibrium. The resultingcomplex mixture was separated by SEC with tryptase SEC buffer withoutheparin and the retention times/volumes and the molecular weights of theindividual protein peaks were determined). The fractions containingprotein were characterized by SDS-PACE to determine the proteincomponents (data not shown). WT tetrameric tryptase had a retention timeof t_(r)=26 min (see for example, peak 1 of FIG. 3A) and a molecularweight of 120 kDa±0.2% according to MALS, which is consistent with thetheoretical molecular weight of 109.537 kDa based on amino acid sequence(excluding glycosylation).

huE104.v1 Fabs formed a homogeneous complex with WT tetrameric tryptasewith a retention time of t_(r)=21.6 mL and a molecular weight of 276.1kDa, as determined by SEC-MALS (data not shown). This would represent acomplex of tryptase tetramer with 4 Fabs bound to it. SDS-PAGE analysisof fractions from this peak confirmed the presence of Fabs and tryptaseprotomers. Thus, huE104.v1 Fabs formed a stable complex with tetramerictryptase and did not affect tetramer stability (data not shown).

Monomeric His6-tagged tryptase (FIG. 3B, run 1) or WT tetramerictryptase (FIG. 3B, run 2) were mixed with 2-fold molar excess ofhuE104.v2 Fab and each complex mixture analyzed individually by SEC. Thefirst protein peak of each chromatogram had a retention time t_(r)=25.8min (FIG. 2B, run 1, peak 2) and t_(r)=26 min (FIG. 2B, run 2, peak 3),respectively. SDS-PAGE analysis of fractions from these two first peaksshowed that both, tryptase and E104.v2 Fab, were present in this peak.Both protein peaks were also analyzed by MALS and a molecular weight of68 kDa±3% was determined, which reflects a complex comprising 1 tryptasemonomer bound to 1 Fab. The second peak of each run had a retention timeof t_(r)=31.6 min min (run 1, peak 6) and t_(r)=31.8 (run 2, peak 7),respectively, and contained only Fab huE104.v2, as determined bySDS-PAGE. The chromatograms of both SEC runs practically superimposed,indicating that huE104.v2 Fab binding dissociates WT tetrameric tryptaseinto monomers, since the retention times were practically identical,independent of whether tetrameric tryptase or His-tagged monomerictryptase was used for complex formation.

When tetrameric tryptase was mixed again with excess huE104.v2 Fab inthe presence of 100 μg/mL heparin and analyzed by SEC in TNH buffer asrunning buffer (run 3), 3 protein peaks were observed: the first peakhas a much shorter retention time (t_(r)=21 min, FIG. 3B, run 3, peak 1)than WT tetrameric tryptase alone (t_(r)=26 min, e.g., FIG. 3A, peak 1).The second peak has a retention time of t_(r)=27.2 min (FIG. 3B, run 3,peak 4) and the last one has a retention time of t_(r)=31.2 min (FIG.3B, run 3, peak 5), which is indicative of Fab alone. SDS-PAGE analysisshowed that both tryptase and Fab were present in peaks 1 and 4, whereinpeak 1 contains the active tetramer bound by huE104.v2 Fabs, and peak 4contains tryptase monomer bound by huE104.v2 Fab (data not shown). Thisled us to conclude that in the presence of 100 μg/mL high concentrationof heparin, only a fraction of tetrameric tryptase was dissociated byhuE104.v2 Fab as in peak 4, while the majority of tryptase remainedtetrameric tryptase bound to hu104.v2 Fabs in peak 1. In summary,huE104.v1 Fab showed no detectable inhibitory activity, while huE104.v2Fab completely dissociated tryptase tetramer. Unlike hu31A.v1 Fab orhuE104.v2 IgG, however, the ability of huE104.v2 Fab to dissociate thetetramer was partially neutralized by high concentration of heparin.Thus, based on these data, we conclude that hu31A.v11 Fab is capable ofdissociating tryptase tetramer with or without high concentration ofheparin, and huE104.v2 Fab is capable of dissociating tryptase tetramerin the absence of high concentration of heparin.

Each tryptase monomer has an identical set of catalytic triad, and thefour monomers assemble to form a tetramer with the four catalytic sitesfacing the middle pore into which the substrate enters. See Pereira etal., supra. Pereira et al. discuss design of small molecule tryptaseinhibitors by blocking the active sites. Several small molecule tryptaseinhibitors in development had been discontinued due to poor selectivityor poor bioavailability. See, e.g., Cairns, J. A., 2005, PulmonaryPharmacology & Therapeutics 18:55-66.

Wild-type tetrameric tryptase is not covalently held together and candissociate into monomers under physiological conditions (Schwartz et al.J. Biol. Chem. 261:7372-7370, 1986; Alter et al. Biochem. J.248:821-827, 1987; Schwartz et al. J. Immunol. 144:2304-2311, 1990). Ithas been reported that mature tryptase demonstrates high enzymaticactivity as a tetramer, and is inactive as a monomer underphysiologically relevant conditions (Schwartz et al., J. Biol. Chem.261:7372-7379, 1986). The data shown herein demonstrate that bothhu31.v11 and huE104.v2 bind to tryptase monomers, dissociate thetetramer to monomers at least at low concentration of heparin, and afterdissociation, remain bound to the monomers. It has also been reportedthat monomeric tryptase may be enzymatically active under certainconditions (Fukuoka et al. J. Immunol. 176:3165, 2006; Fajardo et al.,2003, Biochem. J. 369:603-610). The observations raise the questionwhether a dissociating anti-tryptase antibody will be inferior to anactive site blocking inhibitor such as a tetramer-stabilizing, activesite blocking antibody, because serum inactive monomer may act as amajor sink for the dissociating antibodies that binds to the monomers.And a tetramer-stabilizing, active site blocking antibody may be moreadvantageous as a therapeutic if monomers can be enzymatically activeunder certain conditions.

We first investigated the efficacy of a tetrarer-dissociating antibodyas compared to a tetramer-stabilizing, neutralizing antibody in an insilico experiments using a pharmacokinetic-pharmacodynamic (PKPD) model.The model was developed in Simbiology® software (Mathworks Inc,Cambridge Mass.) to simulate tetrameric tryptase generation,dissociation, and clearance in the circulating and in the lung tissue,as well as the PK and binding effects of the anti-tryptase antibodies.Two types of antibodies are considered: (1) a dissociating antibody thatdissociates the tetramer rapidly upon binding, but also binds monomer;and (2) a stabilizing tetramer-specific antibody, that binds activetetramer only and neutralizes its activity by blocking the access to itssubstrates, but also extends half-life of the tetramer. Atetramer-dissociating antibody with a K_(D) of 0.2 nM was simulatedunder a hypothetical dose regimen of 300 mg administered subcutaneouslyevery four weeks. We assumed a 10% lung partition coefficient of theantibody. For the baseline scenario, we assumed 4 ng/ml total tryptasein the serum and 10 ng/ml total tryptase in the tissue compartment, andfor the high-tryptase scenario, we assumed 10 ng/ml serum tryptase and40 ng/ml tissue tryptase levels. The relative rate constants forphysiologic tetrameric dissociation to monomer and the clearance of eachspecies results in an 8:1 ratio of monomer to tetramer within the totaltryptase pools.

For the simulated dose regimen of 300 mg sc q4w of either dissociatingor stabilizing antibodies, FIG. 4A left panels show the antibodyconcentration of both total antibody and free/unbound antibody in thelung under the baseline scenario, and right panels show thecorresponding lung (free) tryptase levels relative to pre-treatmentlevels for both monomer and tetramer. Results suggest that for adissociating antibody with K_(D)=0.2 nM (top panels), most of the lungantibody remains free (top left panel) indicating adequate drugavailability despite binding to the monomer and tetramer; resultsfurther indicate that the dissociating antibody would achieve asustained reduction of over 90% in tetrameric (active) tryptase (topright panel). For the stabilizing antibody (bottom panels), nearly allof the antibody is free (bottom left panel); however, reduction intetramer is only maintained at or above 80%.

Because the tetramer-specificity of the stabilizing antibody could beconsidered advantageous under high total tryptase conditions, thesesimulations were repeated under the higher tryptase scenario (FIG. 4B).Under these conditions, more dissociating antibody is bound by theinactive monomeric tryptase, resulting in lower free antibody (comparetop left panel of FIG. 4B with that of FIG. 4A) and leading to a lesserbut still good tetramer neutralization (minimum ˜80%) by thedissociating antibody (FIG. 4, top right panel). By comparison, for thetetramer-stabilizing antibody, despite greater free antibody due toabsence of binding to monomer (FIG. 4B, bottom left panel), the antibodyachieves less neutralization (minimum ˜70%) as compared with thedissociating antibody under the same conditions, despite a 10-foldhigher affinity assumed for the stabilizing antibody (K_(D)=0.02 nM) inthe simulations (FIG. 4B, bottom right panel). The lesser neutralizationis due to greater stability (half-life) of the bound target which servesas a “reservoir” for tetramer. Thus, our simulations predict that adissociating antibody is promising and would perform better thantetramer-specific stabilizing antibody under different scenarios tested.

We next tested how the dissociating antibody compares with thetetramer-specific stabilizing antibody at different antibody doses. FIG.4C shows the tetramer neutralization for both types of antibodies, atboth baseline and high tryptase scenarios, as a function of antibodydose level (30, 100, 300 mg so q4w). In the baseline scenario (FIG. 4C,left panels), the dissociating antibody consistently reduces tetrameractivity more than the stabilizing antibody, across all dosesconsidered. In the high tryptase scenario (FIG. 4C, right panels), thedissociating antibody outperforms the stabilizing antibody with respectto tetramer neutralization at all but the lowest dose (30 mg), at whichpoint the two antibodies perform comparably. The generally lesserneutralization achieved using the stabilizing antibody despite a 10×greater affinity (K_(D)=0.02 nM) than the dissociating antibody(K_(D)=0.2 nM), indicates a much higher affinity requirement (>10×) forcomparable tetramer neutralization using the stabilizing antibodycompared to the dissociating antibody. For both scenarios, the optimaldose for (90%) tetramer neutralization is lower for the dissociatingantibody than for the stabilizing antibody (results not shown), despitethe 10× greater affinity assumed for the stabilizing antibody. Thus,under the model, the dissociating antibody would outperform or at leastmatch the stabilizing antibody that has 10× higher affinity, across thedose range and serum and lung tryptase concentrations tested. Inaddition, we demonstrated herein that hu31A.v11 and huE104.v2 IgGcompletely inhibited all tryptase activity. See FIG. 2A and Table 5.

To our knowledge, there has not been any example of tryptase tetramerdissociating antibody developed for therapeutic uses. It is known thatthe inflammatory loci of disease tissues including acute asthmatic lungare acidic as compared to control subjects; see, e.g., Hunt et al. Am.J. Respir. Crit. Care Med. 161:694-699, 2000; Steen et al. J.Neuroscience 15:3982, 1995; Bellocq et al. J. Biol. Chem. 273:5086,1998; and Lardner J. Leukocyte Biol. 69:522, 2001). Published datashowed that the murine monoclonal anti-tryptase antibody B12 neutralizeshuman tryptase beta at neutral pH but is unable to neutralize humantryptase beta activity in an enzymatic assay at acidic pH 6 (see Fukuokaet al. J. Immunol. 176:3165, 2006). Therefore, we tested antibodieshu31a.v11 and huE104.v2 for their ability to inhibit the activity ofhuman tryptase beta in cleaving fibrinogen at both neutral pH and atacidic pH.

Briefly, human tryptase beta tetramer (1.0 μg/mL) was preincubated withthe tested antibody for 30 min in TNH buffer at pH 6.0 or 7.5 (50 mMTris, 150 mM NaCl, 0.1 mg/ml heparin) at room temperature. The mixturewas then incubated with 5 μg human fibrinogen substrate (HaematologicTechnologies, Inc., Cat. No. HIC-0150R) for 2.5 h at 37° C. hu31A.v11Fab, huE104.v2 Fab, and B12 mIgG1 were each tested at 200 μg/mL. Thecleavage products were analyzed by SDS-PAGE and Coomassie Blue staining.

As shown in FIGS. 5A and 5B, human tryptase beta 1 cleaved fibrinogenalpha and beta chains at both pH 6 and 7.5 (compare lane 1, fibrinogenonly, with lane 2, fibrinogen plus tryptase beta 1). Antibody hu31A.v11Fab inhibited human tryptase beta at both pH 6 and pH 7.5 (lane 3),while huE104.v2 Fab, under the assay condition of 0.1 mg/ml of highconcentration of heparin, did not (lane 5). Lane 6 was B12 mIgG1 alone.The decrease in fibrinogen alpha chain is indicative of tryptaseproteolytic activity. The fibrinogen beta chain is also cleaved, but thebeta chain cleavage tends to be obscured on the gel. The intensity ofthe alpha chain was thus quantified and shown in the bottom panels ofFIGS. 5A and 5B. Thus, hu31A.v11 Fab inhibited tryptase activity at pH 6and 7.5, while huE104.v2 Fab under the assay condition of high heparinconcentration, was not inhibitory.

The inhibitory activity of IgG format antibodies (hu31A.v11 IgG4 andhuE104.v2 IgG4) was also evaluated using S-2288 peptide as a substratein an inhibition assay in the presence of 1 nM tryptase and 400 pMchromogenic S-2288 in TNH buffer at pH 6, 7, or 8. The finalconcentration of heparin in this experiment was about 95 μg/ml. Bothhu31A.v11 and huE104.v2 in the IgG format completely inhibited tryptaseactivity at pH 6, 7, and 8 (data not shown).

Therefore, hu31A.v11 IgG and huE104.v2 IgG both bind to tryptase withhigh affinity and also efficiently inhibit tryptase activity underphysiologically relevant conditions for tryptase-associated disorderssuch as asthma. Interestingly, in our experimental conditions, murinemonoclonal anti-human tryptase antibody B12 IgG1 also showed inhibitionof human tryptase beta at pH 6 (FIG. 5A, lane 4), contrary to publishedresults. The discrepancy may be attributed to any residual non-tryptaseprotease activity present in the material used for the assay in thepublished data that was activated by acidic pH and resistant to B12inhibition.

Example 3: Structural Analysis of Anti-Tryptase Antibodies

A. 31A Family Antibodies

(i). X-Ray Crystallography

Wild-type tetrameric tryptase was mixed with 1.5-fold molar excess Fabhu31A.v11 and 2-fold molar excess soybean trypsin inhibitor (STI)(Roche) and incubated for 10 min at room temperature. STI was added tofacilitate crystallization. The mixture was then subjected to SEC usingan S200 column (GE Healthcare) in 10 mM MOPS (pH 6.8), 0.5 M NaCl.Fractions containing the ternary complex of tryptase, STI, and Fabhu31A.v11 were pooled and concentrated to 40 mg/mL.

Crystals of tryptase/Fab hu31A.v11/STI were grown at 19° C. using thevapor diffusion method in hanging drops. Crystallization buffercontaining 0.1 M Tris (pH 7.5), 0.2 M lithium sulfate, and 5%polyethylene glycol (PEG) 4000 was mixed in equal volume with theprotein solution. The crystals were dipped in artificial mother liquorcontaining 25% ethylene glycol and vitrified in liquid nitrogen.

Table 7 shows X-ray data collection and refinement information for thestructure of Fab hu31A.v11/tryptase/STI. Diffraction data extending to2.15 Å were collected in a hexagonal lattice at ALS Beamline 5.0.2. Datareduction and scaling allowed assignment of the Laue class to 6/mmm(Otwinowski, Methods in Enzymol. 276, 307-326, 1997; Winn et al. ActaCrystallogr. Sect. D-Biol. Crystallogr. 67, 235-242, 2011). Based onunit cell volume and proposed protein content, a single Fabhu31A.v11/tryptase/STI complex was expected in the crystallographicasymmetric unit. The structure was solved using molecular replacement(McCoy et al. J. Appl. Crystallogr. 40:658-674, 2007) in space groupP6₂22. The following search probes were used, each as separate bodies: apreviously determined tryptase protomer derived from PDB accession 4A6L(Liang et al. Bioorg. Med. Chem. Lett. 22:1049-1054, 2012), STI from PDB1AVU (Song et al. J. Mol. Biol. 275:347-363, 1998), an Fv fragmentstripped of CDR loops from PDB 1FVC (Eigenbrot et al. J. Mol. Biol.229:969-995, 1993), and the constant region from PDB 1FVD. After somerestrained refinement (Murshudov et al. Acta Crystallogr. Sect. D-Biol.Crystallogr. 67:355-367, 2011), electron density maps permittedcorrection of Fab protein sequence and fitting missing residues and sidechains into clear density (Emsley et al. Acta Crystallogr. Sect. D-Biol.Crystallogr. 66:486-501, 2010). Waters were added automatically (Adamset al. Acta Crystallogr. Sect. D-Biol. Crystallogr. 66:213-221, 2010).More rounds of model adjustments and refinement (BUSTER software; GlobalPhasing Ltd., 2011) led to the final model. The model is continuous fortryptase residues Ile16-Lys244 (chymotrypsinogen numbering), STIresidues Asp1-Asp177, Fab light chain residues Asp1-Cys214 (Kabatnumbering) (Kabat, Sequences of Proteins of Immunological Interest.Bethesda, Md., U.S. Dept. of Health and Human Services, Public HealthService, National Institutes of Health, 1991). The Fab heavy chain iscontinuous except for a five amino acid residue gap in the constantdomain. The value for the shape complementarity statistic (Sc) (Lawrenceet al. J. Mol. Biol. 234:946-950, 1993) is 0.73, consistent with othertight-binding Fab/antigen complexes. The largest inter-protein contactarea is that between tryptase and STI, as they each lose 1260 Å² solventaccessible surface (Broger C, xsae, F. Hoffman-La Roche, Basel,Switzerland, 2000) at their interface. In contrast, the Fab/tryptasecontact is only 760 Å² on each side, evenly distributed between lightand heavy chains of the Fab. Crystal packing contacts 4 Å or less arewell distributed around STI, tryptase, and all four Fab domains, andinclude numerous hydrogen bonds as well as hydrophobic contacts.

TABLE 7 X-ray Data Collection and Refinement Fab hu31A.v11/Tryptase/STIData reduction X-ray source ALS 5.0.2 Wavelength (Å) 0.97395 Resolutionrange (Å) 31.48-2.15 (2.226-2.149) Space group P₆222 Unit cell edges (Å)128.52 128.52 245.88 Unit cell angles (°) 90 90 120 Total reflections308799 Unique reflections 65702 (6404) Multiplicity 4.7 (4.8)Completeness (%) 99.8 (100) Mean I/sigma(I) 14.2 (2.3) Wilson B-factor(Å²) 37.02 R-symm 0.086 (0.681) Refinement Refs for R-free 1996 R-work0.190 R-free 0.233 Number non-H atoms 7057 macromolecules 6608 ethyleneglycol 8 water 441 Protein residues 850 RMS(bonds) 0.010 RMS(angles) 1.1Rama favored (%) 97 Ave. B-factor (Å²) 45.7 macromolecules 45.6 ethyleneglycol 59.7 solvent 47.1

(ii) Epitope Mapping of Anti-Tryptase Antibodies Analyzed byHydrogen-Deuterium Exchange (HDX) and Measured by MS

Deuterium uptake rates of monomeric tryptase in the presence and absenceof antibody were measured to determine structural regions that aremodified upon antibody binding. Bound samples contained a 1:1 mixture oftryptase and the respective antibody, prepared and incubated at roomtemperature for 1 h. Tryptase concentration prior to deuterium labelingwas 30 pM in antibody-bound and unbound samples. HDX experimentsinvolved diluting samples 15-fold into deuterium labeling buffercontaining 20 mM histidine acetate at pD 7.0. Six labeling times,logarithmically sampled between 30 sec and 1000 min, were taken intriplicate, quenched by lowering the pH to pH 2.5 and adding 2 Mguanidinium chloride (GdmCl) and 0.25 M tris(2-carboxyethyl)phosphine(TCEP), and injected into a cold online system, as previously described(Mayne et al., J. Am. Soc. Mass. Spectrom. 22:1898-1905, 2011).

Briefly, samples were first passed through an immobilized pepsin column(2.1×30 mm, Applied Biosystems) and loaded onto a trap column (AcquityVanguard C) for desalting. Peptide fragments were then separated byreversed-phased chromatography using an Acquity UPLC™ BEH C₁₈ column(1.7 μm particle size, 1.0×50 mm) and introduced into the massspectrometer (Thermo Orbitrap Elite™, 120 k Hz resolution at m/z 400)for mass analysis. Chromatographic mobile phases were prepared aspreviously described to minimize deuterium back exchange (Walters etal., J. Am. Soc. Mass Spectrom. 23: 2132-2139, 2012). The ExMS program(Kan et al. J. Am. Soc. Mass. Spectrom. 22:1906-1915, 2011) was used toidentify deuterated peptides and prepare extracted ion chromatograms,which were then analyzed by in-house python scripts (Walters et al.Proc. Natl. Acad. Sci. USA 110:18898-18903, 2013). These scripts combinedegenerate charge states, fit isotopic distributions with binomials, andextract the number of deuterium carried, on average, by each peptide.

(iii) Intact Mass by Liquid Chromatography/Mass Spectrometry (LC/MS)

Purified proteins (including tryptase and mutants thereof) wereacidified with 0.1% trifluoroacidic acid (TFA) (Thermo, Rockford, Ill.)and diluted to a final concentration of 10 pM. Samples were analyzed onan Agilent 6520 Accurate-Mass quadrupole time-of-flight (Q-TOF) coupledwith an Agilent 1260 Infinity high performance liquid chromatography(HPLC)-Chip Cube Interface (Agilent, Santa Clara, Calif.). Proteins wereseparated by reversed phase HPLC on a PLRP-S 150 mm×75 μm column at 40nL/min flow rate with a 10 min, 5-80% gradient of aqueous solvent A (97%H20, 3% acetonitrile, 0.1% formic acid) to organic solvent B (98%acetonitrile, 0.1% formic acid). Data was collected with a MassHunter®Workstation (Agilent, Santa Clara, Calif.) and raw mass spectra weredeconvoluted to generate intact mass data. Major peaks were observed at61764.4 Da and 63152.9 Da. The addition of multiple GlcNAc (203 Da)masses were also observed.

(iv) Results

The crystal structure of human tryptase shows that each monomer of thetetramer contacts its neighbors at two different interfaces through sixloop segments: the large interface (between protomers A/D and B/C) orthe small interface (between protomer A/B and C/D) according to protomernomenclature described by Pereira et al. Nature 392:306-11, 1998, whichis incorporated herein by reference in its entirety. The six surfaceloops of each protomer surround the active site and engage inintermonomer contacts (Pereira, supra). Among which, the 147 loop, the70-80 loop and the 37 loop engage in the interaction of small interface,and the 173 flap, the 97 loop and the 60 loop are important in largeinterface interaction. While the small interface involves onlyhydrophobic interactions, the large interface comprises several polar,charged interactions in addition to hydrophobic interactions. Heparinfurther stabilizes the small interfaces, which are believed to be theshear points of the tetramer, by binding non-covalently to positivelycharged residues spanning the two adjacent protomers A/B and C/D. SeePereira supra. Naturally, the small interfaces are the shear points ofthe tetramer, followed by the dissociation of the dimer held by thelarge interfaces. The half-life of tryptase is about 2-3 hours incirculation in human blood after development of systemic anaphylaxis(see, e.g., Schwartz et al. J. Cin Invest. 83:1551-1555,1989). Thenormal half-life of the tetramer is about 30 minutes.

We determined whether the small or the large protein interfaces thathold the tryptase tetramer together are destabilized upon hu31A.v11 orhuE104.v2 binding to tryptase. Two tryptase mutants were generated whichresulted in covalent linkage of two neighboring protomers in thetetramer via an intermolecular disulfide bond in either the largeinterface (between protomers A/D and B/C) or the small interface(between protomer A/B and C/D) according to protomer nomenclaturedescribed by Pereira et al. supra. Thus, when dissociation of the smallinterface is prevented due to covalent disulfide-linked dimer formation,dissociation of the large interface is still permitted. Conversely, whendissociation of the large interface is prevented due to covalentdisulfide-linked dimer formation, dissociation of the small interface isstill permitted.

Tyr75 in the small interface and Ile99 in the large interface(chymotrypsinogen numbering, FIG. 7) were individually mutated tocysteine. These sites were selected because they face themselves inthese interfaces due to the quasi 2-fold symmetry of the tetramer(Sommerhoff et al. Proc. Natl. Acad. Sci. USA 96:10984-91, 1999).Replacement of Tyr75 or Iie99 to cysteine in silico using PyMOL softwareshowed respective distances of 2.4 Å and 3.2 Å between the thiols ofeach opposing cysteines, which is somewhat greater than a typicaldisulfide bond length of 2.05 Å. Nonetheless, the two resulting tetramermutants were expressed and purified and contained a disulfide bond thathad formed between the respective interfaces as determined by trypticpeptides MS analysis. Further, both mutants were also enzymaticallyactive, but with about half the catalytic efficiency (k_(cat)/K_(M))when compared to wild-type tetrameric tryptase (Table 8).

TABLE 8 Enzymatic activity of wild-type and mutant tryptase CatalyticK_(M) V_(max) k_(cat) k_(cat)/K_(M) efficiency Tryptase (μM) (nM/sec)(1/s) (1/(M*s) (Mutant/WT) WT 554 400 100 180505.42 1 Y75C 320 133 33.25103906.25 0.57 Small interface locked I99C 798 235 58.75 73621.55 0.41Large interface locked

The size of these mutants complexed with hu31A.v11 Fab or huE104.v1 Fabwas analyzed. Tryptase mutants Y75C and I99C were each mixed with a2-fold molar excess of Fab hu31A.v11 for complex formation, as describedabove for wild-type tryptase, and analyzed by size exclusionchromatography. As a reference for comparison, wild-type tetramerictryptase was complexed with a Fab from the antibody huE104.vi, whichbinds specifically to tryptase, but does not dissociate the tetramer,and which loses inhibitory activity at high antibody to tetramer ratio.Wild-type tetrameric tryptase complexed with huE104.v1 Fab had aretention time of 21.6 min (FIG. 6A, reference peak). SEC-MALS analysisof this complex determined a molecular weight of 276.1 kDa, indicativeof a complex comprising one tryptase tetramer with four bound Fabs.

Fab hu31A.v11 formed complexes with tetrameric tryptase mutants Y75C andI99C with retention times of 25.6 min (FIG. 6A, run 2, peak 1) and 23.9min (FIG. 6A, run 3, peak 2), respectively (FIG. 6A). Both retentiontimes are longer than that of the reference complex, indicating thatboth tetramer mutants dissociate into their respective covalently linkeddimers upon complex formation with Fab hu31A.v1. Samples from each peakwere collected and analyzed by SDS-PAGE to confirm the species in eachpeak (FIG. 6B). SDS-PAGE analysis of these eluted protein peaks showthat both Fab hu31A.v1 and respective tryptase dimers were present inthe same fraction (FIG. 6B). Peak 3 (T_(r) of 28.1 min) and peak 4(T_(r) of 31.6 min) of run 4 represented WT monomer complexed with Fabhu31A.v11 and excess Fab, respectively. A small amount of peak 3 samplewas carried over to peak 4 sample in SDS PAGE. Both tryptase mutants incomplex with Fab hu31A.v11 run with a longer retention times than thereference peak, indicating that both the large and the small interfaceare destabilized upon Fab hu31A.v11 binding. This destabilizationresults in tetramer dissociation, as observed for wild-type tryptasetetramer, which ultimately inactivates the proteolytic activity oftryptase. B12 Fab also destabilized both mutant tetramers (data notshown).

The binding of the humanized anti-tryptase antibody hu31A.v11 to maturehuman tryptase beta 1 was studied using X-ray crystallography todetermine the structure of the molecular complex between tryptase andthe Fab fragment of hu31A.v11 at 2.15 Å resolution. The result was acrystallographic asymmetric unit containing one tryptase/STI (soybeantrypsin inhibitor) complex interacting with one hu31A.v11 Fab. STI wasincluded for the purpose of crystallization.

One definition of epitope is the set of tryptase amino acids that arewithin 4 Å of any atom of the hu31A.v1 Fab. The program PyMOL was usedto find epitopes. The amino acid residues in the tryptase polypeptidechains can be numbered according to a convention (chymotrypsinogennumbering) that deviates from sequential numbering to permit comparisonsacross homologous proteins. A table translating between this tryptaseresidue numbering scheme and the simple sequential scheme is provided(FIG. 7). Tryptase residues below are named according to the convention,with the gene-sequential numbering scheme in parenthesis.

The epitope of hu31A.v11 on tryptase includes the following residues:H36, 050, V60c, K60d, D60e, L61, A62, A63, R65, P84, V85, S86, R87,E109, E110, P111 (chymotrypsinogen numbering, corresponding to H51, 067,V80, K81, D82, L83, A84, A85, R87, P103, V104, S105, R106, E128, E129,and P130, respectively, of SEQ ID NO: 71). See FIG. 8. Residues of the60s, 80s, and 100s loops of tryptase are in intimate contact with Fabhu31A.v1, while His36 and Gln50 residues are more on the periphery ofthe interaction with the Fab. See also Pereira et al. supra. TheFab-tryptase contact excludes 760 Å² from solvent (each side), withequal contributions from the Fab light chain (Va30, Thr31, Tyr32, Tyr34,Arg50, Tyr90, His92, Ser93, Tyr94) and heavy chain (Phe50, Ser52, Gly53,Ser54, Ser55, Thr56, Tyr58, Arg95, Tyr97, Asp98). It is considered thatthe epitope residues described above would also apply to other31A-derived antibodies.

When the Fab/tryptase dimer from the ternary complex was superposed ontotryptase protomers A and D from the wild-type tetramer (Pereira et al.supra), we found that the two Fabs sit on the same side of the tetramer,almost perpendicular to the tetramer plane (FIG. 9). Repeating thisprocess for protomers B and C places two Fabs on the opposite side ofthe tetramer. Notably, these results highlight the steric clashesbetween the Fab light chains, consistent with the fact that the Fabelutes as a 1:1 complex with monomeric tryptase on SEC and not as acomplex with tetrameric tryptase (FIG. 9).

Tetrameric tryptase is not covalently held together and can dissociateinto monomers under physiological conditions, as monitored by the decayin the enzymatic activity over time and changes in the circulardichroism (CD) spectrum in solution (Schwartz et al. J. Biol. Chem.261:7372-7379, 1986; Schwartz et al. J. Immunol. 144:2304-11, 1990).Binding of hu31A.v11 (Fab or IgG) to each protomer of the tetramer wouldpromote and accelerate the dissociation of the tetramer and prevent anytetramer re-association due to the steric clashes of the Fabs when boundto tryptase. Thus, tryptase dissociates and becomes enzymaticallyinactive monomer in a more rapid fashion due to allosteric changes oneach protomer caused by hu31A.v11 binding. Because of the pseudo-2-foldsymmetry in the tetramer, almost all of the interactions that constitutethe small and the large interfaces exist twice. This also means thateach subtle change in the tryptase structure caused by hu31A.v11 bindingoccurs twice in each interface, thereby potentiating destabilization.

The complex structure of Fab hu31A.v1 with tryptase shows significantchanges in the 60s loop, which is in the large interface. In particular,when bound by hu31Av1 Fab, residue Val60c (corresponding to Val80 of SEQID NO: 71) has a significant shift in the side chain when compared toits position found in the unbound tryptase structure. In the unboundtryptase tetramer, Tyr173d from the neighboring protomer sits in ahydrophobic pocket created by residues Val60c and Val90 (FIG. 10, bothare by chymotrypsinogen numbering, see also FIG. 7). In the antibodycomplex, the conformational change of Val60c creates steric hindrancethat prevents Tyr173d binding to that pocket. Since binding of hu31A.v1to tryptase involves interactions with parts of the 60s loop and nearbyresidues, it may be responsible for this conformational change inVal60c. This could lead to destabilization of the large interface andwould thus enhance dissociation of tryptase tetramer into inactivemonomers.

The imidazole ring of His36 (chymotrypsinogen numbering; correspondingto His51 of SEQ ID NO: 71) in tryptase makes hydrophobic interactionswith Fab hu31A.v1, resulting in changes the Ca trace in the 30s loop oftryptase residues His36, Pro37a, and Tyr37b when compared to tryptaseprotomers in the unbound tetramer structure. This affects theconformation of the Tyr37b side chain, such that a key hydrophobicinteraction with a neighboring protomer comprising Pro152 and Pro152a inthe small interface may be weakened (FIG. 11, all are bychymotrypsinogen numbering, see also FIG. 7).

The complex formation studies of the two different disulfide-lockedtryptase variants Y75C and I99C with hu31A.v1 Fab show that both thelarge and the small interface of tetrameric tryptase are destabilized bythe antibody. Both the steric clashes of Fabs bound to tetramer andconformational changes to key interacting residues of the large and thesmall interface are likely important in contributing to tetramerdissociation. These two factors are not mutually exclusive. Sterichindrance stemming from hu31A.v11 Fabs when bound to each protomer ofthe tetramer demonstrated in silico indicate that two Fabs from a singleIgG could typically never bind simultaneously to one tetramer (FIG. 9).This observation is consistent with the finding that both the Fab andthe IgG of hu31A.v11 are capable of dissociating the tetramer, therebyinhibiting enzymatic activity. This observation also indicates thatdissociated monomers bound by the antibody are unlikely to reassemble toform a tetramer.

Next, HDX experiments of monomeric tryptase bound to hu31A.v11 Fab wereperformed to study the binding interactions in solution. The degree ofHDX over time was monitored by mass spectrometry. While this methodprovides information indicative of the binding epitope of hu31 Å.v1, italso detects allosteric changes in conformational stability that couldoccur away from the antibody binding epitope. Amide bonds that showed aslower exchange in tryptase when hu31A.v11 was bound were located in theregions of residues 25-29, 40-41, 57-59, 60-61, 66-67, 83-88, 108-110,and 231-233 (chymotrypsinogen numbering). When comparing these regionswith tryptase residues within 4 Å of the Fab hu31A.v11 as seen in thecrystal structure, a high degree of overlap between both methodsidentifying binding epitope residues in the 60s, the 80s and 100s-loopwas observed (FIG. 8). Other areas identified by HDX such as positionsin the 20s, 40s, 50s, and 230s regions of the primary tryptase sequenceare not in direct contact with the antibody according to the crystalstructure, but show a reduction in conformational dynamics in thepresence of hu31A.v11. Interestingly, residues 57-59 seem to beallosterically affected by hu31A.v11 binding. This short a-helixcontains the residue His57 (chymotrypsinogen numbering) that is part ofthe catalytic triad in serine proteases and essential for catalyticactivity. Although residues 40 and 41 identified by HDX are not indirect contact with hu31A.v11, they are in an intriguing structurallocation as part of an anti-parallel beta-sheet displaying Tyr37b(chymotrypsin numbering) in their hairpin loop, which is an importantcontact residue for the small interface as discussed above (FIG. 11).Structural alterations to this area could influence the stability of thesmall interface and potentially lead to tetramer dissociation.

Peptide bonds in the 20s loop (all residues in the unstructured loop)and 230s region (also referred to as 230s helix), which also undergochanges in their structural dynamics according to HDX, are far away fromthe binding site of hu31A.v11. HDX experiments monitor changes instructural dynamics, and do not distinguish between changes in bulkconformation versus changes in the energetic stability of a particularconformation. Overall, we found a high degree of consistency between thestructural information obtained by HDX and X-ray crystallography andidentified additional residues that are allosterically affected byhu31A.v11 binding.

B. Structural Analysis of E104-Derived Anti-Tryptase Antibodies

(i) Materials and Methods

huE104.v1 Fab was used to generate crystal structure with tryptasetetramer because binding of huE104.v1 does not dissociate the tetramer.Tryptase/huE104.v1 crystals were grown at 19° C. using the vapordiffusion method by mixing protein in 1:1 (v/v) with a reservoirsolution containing 0.1 M Tris (pH 8.5), 0.2 M calcium chloride, 20%polyethylene glycol (PEG) 4000 and 8% pentaerythritol ethoxylate. Thecrystals were cryo-protected in artificial mother liquor containing 0.1M Tris (pH 8.5), 0.2 M calcium chloride, 35% PEG 3350 and flash frozenin liquid nitrogen. Diffraction data were collected at SSRL beamline12-2 in a monoclinic lattice extending to 3A resolution using a Pilatus6M pixel array detector and 0.9795 Å wavelength X-rays. Data werereduced (Kabsch, Acta Crystallogr. D Biol. Crystallogr. 66:125-132,2010; Vonrhein et al. Acta. Crystallogr. D67:293-302, 2011) and scaled(Winn et al. Acta Crystallogr. D Biol. Crystallogr. 67:235-242, 2011),and the structure solved by molecular replacement (McCoy et al. J. Appl.Crystallogr. 40:658-674, 2007) in space group P21 revealing a tryptasetetramer bound by four Fabs. The molecular replacement search probeswere a tryptase protomer from PDB accession 4A6L and an antibody Fabfragment derived from PDB accession 1FVD by scanning modified versionswith a range of elbow angles using the rotation function only. Afterlimited refinement, one Fab constant region was replaced in a molecularreplacement search using just the constant region from 1FVD. Tryptaseresidue numbering was changed to a chymotrypsinogen scheme andFab-E104v1 residue numbering to the Kabat scheme. Model and electrondensity map inspection and adjustments were performed using Coot (Emsleyet al. Acta Crystallogr. D Biol. Crystallogr. 66:486-501, 2010) and thestructure refined using REFMAC5 (Murshudov et al. Acta Crystallogr. DBiol. Crystallogr. 67: 355-367, 2011) and Phenix.refine (Adams et al.Acta Crystallogr. D Biol. Crystallogr. 66, 213-221, 2010). Datacollection and refinement metrics appear in Table 9. HDX experimentswere performed as described above.

TABLE 9 Table of X-ray metrics for tryptase beta-huE104.V1 Fab DataX-ray source SSRL 12-2 Wavelength (Å) 0.9795 Res. range (Å) 50 −3.0(3.112 −3.005) Space group P21 cell a, b, c (Å) 89.573 168.811 114.652cell α, β, γ (°) 90 109.97 90 Total reflections 214206 Uniquereflections 62353 Multiplicity 3.4 (3.5) Completeness (%) 97.8 (99.7)Mean I/σ(I) 11.3 (2.1) Wilson B (Å²) 72.2 R-symm 0.098 (0.577)Refinement Reflections for R-free 1266 R-work 0.187 (0.278) R-free 0.232(0.340) Number non-H atoms 20794 macromolecules 20689 ligands 100 ions 3water 2 Protein residues 2702 RMS(bonds) (Å) 0.01 RMS(angles) (°) 1.2Ramachandran favored(%) 94 Ave B-factor (Å²) 71.5 macromolecule 71.6ligands 48.4 ions, water 71.6

(ii) Results

In order to determine if the small or the large protein interfaces thathold the tryptase tetramer together are destabilized upon huE104.v2 Fabbinding, tetrameric tryptase mutant Y75C or I99C (see above) were mixedwith a 2-fold molar excess of Fab huE104.v2 for complex formation andanalyzed by SEC (FIG. 6C). huE104.v2 Fab formed complexes withtetrameric tryptase mutant Y75C and the chromatogram shows two peakswith retention times of t_(r)=21.6 min (FIG. 6C, run 1, peak 1) andt_(r)=31 min (peak 4) comprising excess Fab. huE104.v2 Fab formedcomplexes with tetrameric tryptase mutant I99C and the chromatogramshows two peaks with retention times of t_(r)=23.8 min (FIG. 6C, run 2,peak 2) and t_(r)=31 min (run 2, peak 5) comprising excess Fab. SDS-PAGEanalysis showed that both huE104.v2 Fab and tryptase dimer mutant werepresent in peaks 1 and 2 and excess Fab was present in peaks 4 and 5(data not shown). For comparison, huE104.v2 Fab in complex with WTtryptase tetramer had a retention time of t_(r)=26 min (FIG. 6C, run 3,peak 3), comprising monomer bound by Fab huE104.v, that is smaller thantryptase tetramer mutant I99C in complex with huE104.v2 (t_(r)=23.8 min,run 2, peak 2). The tryptase mutant Y75C (the small interface-lockedmutant tetramer) in complex with huE104.v2 Fab had a similar retentiontime as the stable, intact complex of WT tryptase tetramer bound to fourFabs of huE104.v1 (see FIG. 6A, run 1, ref peak). On the other hand,tryptase mutant I99C (the large interface-locked mutant tetramer) incomplex with huE104.v2 had a longer retention time indicative of asmaller complex than the first peak in run 1. The results indicate thathuE104.v2 Fab was only able to dissociate the small interface, but notthe large interface of the tetramer. Thus, when binding to wild typetryptase tetramer, huE104.v2 Fab binding dissociates the small interfaceonly. Under the experimental conditions, the dissociated small interfacemay be quickly restored by heparin present at a high local concentration(e.g., 0.1 mg/ml), which would lead to the reassembly of the tetramer.This heparin concentration is substantially higher than physiologicalheparin concentration, which has been reported to be about 1.5 μg/ml ofserum (see, e.g. Engelberg et al., Circulation 23:578-581, 1961 andDavids et al. S. Afr. Med. J. 100:307-307, 2010). At low concentrationof heparin, huE104.v2 Fab is capable of completely dissociating WTtryptase tetramer and neutralizing tryptase activity, though less potentthan huE104.v2 in the IgG format.

To gain further insight into the exact binding epitope of huE104.v2 ontryptase, we attempted to crystallize the Fab huE104.v2/tryptasecomplex. Since we could not obtain any suitable crystals, the complex ofWT tetrameric tryptase bound to four Fabs E104.v1 isolated by sizeexclusion chromatography was used for crystallization. huE104.v1 andhuE104.v2 differ only in two vernier framework positions, while the HVRsare identical. Thus huE104.v1 was a good surrogate to elucidate thebinding epitope of huE104.v2. The binding of the humanized anti-tryptaseantibody huE104.v1 (see Example 1) to mature human tryptase beta 1 wasstudied using X-ray crystallography to determine the structure of themolecular complex between tryptase and the Fab fragment of huE104.v1 at3.0 angstrom (Å) resolution. The result was a crystallographicasymmetric unit containing one tryptase tetramer, stabilized bycomplexing with EGR-chloromethylketone interacting with four huE104.v1Fabs in such a way that each Fab interacts almost exclusively with onlyone of the tryptase protomers (FIG. 12A). The intermolecular crystalpacking environment does not include large voids, and has a moderatenumber of crystal packing contacts of less then 4 Å. For instance, twohuE104.v1 Fab VL domains experience a non-crystallographic 2-foldcontact at their ABDE β-strands face, involving H-bonds from Thr and Serchain chains and others. Another smaller region with severalintermolecular contacts exists between a heavy chain constant domain(near the peptide link to the same chain's variable domain) and the“bottom” of an adjacent light chain constant domain. There are moreresidues in packing contacts for the Fabs (average 12) than for thetryptase protomers (average 3.5), despite the tryptase protomers havingmore residues (243 versus approximately 215). In this sense, thecrystals are formed at least in part via Fab-Fab intermolecularcontacts.

Three copies of huE104.v1 Fab displayed closely similar elbow angles(the angles between the variable domains (VH and VL) and constantdomains (CH and CL) of 140°, 138° and 141°. The fourth copy of huE104.v1Fab was characterized by a relative poor electron density for itsconstant region and displayed an elbow angle of 152°. Antibodyantigen-binding surface areas (paratopes), calculated as solventaccessible surface area lost (Adams et al. Acta Crystallogr. D Biol.Crystallogr. 66:213-221, 2010) to the contact with a tryptase protomer,average 682 Å² and are dominated by the heavy chain, 71% versus 29% forthe light chain. The shape complementarity statistic (Lawrence et al. J.Mol. Biol. 234:946-950, 1993), Sc, averaged 0.76, which is on the highend for antibodies with protein antigens. The resolution of our resultwas too low to discern water structure, but the Sc values suggest thereare likely very few interfacial waters. Use of non-crystallographicsymmetry (NCS) restraints produced superior refinements according to thevalue of R-free. The root-mean-squared deviations (RMSDs) forsuperposition of Ca atoms of Fab domains (VL, CL, VH or CH1) were on theorder of a few tenths of an angstrom.

Antibody residues within 4 Å of their partner tryptase were closelysimilar for the four copies and include: light chain residues Y29, N30,R32 (HVR-L1), R94 (HVR-L3) and heavy chain residues G31, Y32 (HVR-H1),S52, S53, A54, T56, F58 (HVR-H2) and P96, R97, G98, Y99, R100e (HVR-H3).

Each huE104.v1 Fab contacts a closely analogous region of a partnertryptase protomer. This epitope is approximately 90° away from theactive site substrate binding cleft, and places each Fab projectingperpendicular from the plane of the roughly square planar tryptasetetramer. Since the tryptase protomers associate in an up/down/up/downmanner around the tryptase ring, there are two Fabs projecting “up” fromthe tetramer and two Fabs projecting “down” (FIG. 12A).

The crystallographic asymmetric unit (asu) includes 4 tryptase protomersorganized into an approximately symmetric tetramer. The four tryptaseprotomers each are covalently modified at the active site (fromtreatment using Glu-Gly-Arg-chloromethyl ketone) and superpose withpairwise rmsd based on Ca atoms of about 0.1 Å. If each protomer werewithin 4 Å of only one of the four Fabs in the asu, then it would bepossible to define four huE104.v1 epitopes, one for each tryptaseprotomer, and, lacking strict symmetry, these four epitopes might differslightly. There are a few tryptase residues that are within 4 Å of a Fabthat does not provide the bulk of interactions with it, referred toherein as a “non-partner Fab.” Since this is true, it is also possibleto define the huE104.v1 epitope for the tryptase tetramer.

The program PyMOL was used to identify the epitope, which in thisExample is the set of tryptase amino acids that are within 4 Å of anyatom of the huE104.v1 Fab. The amino acid residues in the tryptasepolypeptide chains can be numbered according to the chymotrypsinogennumbering scheme, as described above (FIG. 7). Tryptase residues beloware named according to the chymotrypsinogen numbering scheme, with thegene-sequential numbering scheme in parenthesis.

There are 4 epitopes defined between a tryptase protomer and its partnerFab. All four contain a very nearly identical set of tryptase amino acidresidues. Those residues are: W38, Q50, D60e, L61, A62, R65, Q81, L82,L83, P84, V85, S86, R87, E107, L108, E109, and E110 (chymotrypsinogennumbering, corresponding to residues W55, Q67, D82, L83, A84, R87, Q100,L101, L102, P103, V104, S105, R106, E126, L127, E128, and E129,respectively, using the gene-sequential numbering scheme, with residuescorresponding to positions of SEQ ID NO: 71). Residue L61 (L83 of SEQ IDNO: 71) is absent for two of the four, but only barely exceeds the 4 Åcriterion. Residue Q81 (Q100 of SEQ ID NO: 71) is absent for one of thefour. If this were not the case, then all four would be identical by thedefining criterion.

Also, non-partner Fabs make a small number of 4 Å contacts in thetetramer. It is these tryptase residues that distinguish a tetramerepitope from a protomer epitope. The contacted tryptase residues are:Q20 and R187 (Q35 and R216, respectively, of SEQ ID NO: 71).

Thus, the epitope of huE104.v1 on the tryptase tetramer is the sum of: 4instances of each of the following: W38, Q50, D60e, L61, A62, R65, L82,L83, P84, V85, S86, R87, E107, L108, E109 and E110 (chymotrypsinogennumbering, corresponding to residues W55, Q67, D82, L83, A84, R87, L101,L102, P103, V104, S105, R106, E126, L127, E128, and E129, respectively,of SEQ ID NO: 71), plus three instances of Q81 (Q100 of SEQ ID NO: 71),plus 1 instance of Q20 (Q35 of SEQ ID NO: 71), plus three instances ofR187 (R216 of SEQ ID NO: 71). It is considered that the epitope residuesdescribed above would also apply to other E104-derived antibodies,including huE104.v2.

Although huE104.v1 and v2 have the same CDR sequences, and would bind tothe same epitope, the two variants behave differently in Fab as well asin IgG: huE104.v2 Fab dissociates the tetramer and more specifically atthe small interface, while huE104.v1 Fab does not; and in IgG, huE104.v2dissociates and inactivates the tetramer, while huE104.v1 loses theinhibitory activity at high antibody to tryptase ratio. We hypothesizedthat although huE104.v1 and huE104.v2 bind to the same contact residueson tryptase, subtle differences exist between how they interact withtryptase. In order to identify differences in the interaction betweenhuE104.v1 and huE104.v2 Fabs with tryptase in solution, HDX experimentswere performed with monomeric tryptase alone or bound to eitherhuE104.v1 or huE104.v2, and the degree of hydrogen-deuterium exchangeover time was monitored by mass spectrometry. While this method providesinformation indicative of the binding epitopes of huE104.v1 andhuE104.v2, it overall monitors changes in structural dynamics that couldoccur away from the antibody binding epitope. These measurements do notdistinguish between changes in bulk conformation versus changes in theenergetic stability of a particular conformation.

Amide bonds that showed a slower exchange in tryptase when eitherhuE104.v1 or huE104.v2 were bound were located to the regions ofresidues 25-27, 60-61, 66-68, 88 and 108-110 (chymotrypsinogennumbering; Table 10), but amide bonds that showed slower exchange thatwere specific to only huE104.v1 or huE104.v2 were also found. Forexample, amide bonds of tryptase residues 47-50, 54-55, 85-87, 119-122,179, 230-231, and 244-245 (chymotrypsinogen numbering) showed slowerexchange only when huE104.v1 was bound. On the other hand, amide bondsof tryptase residues 25, 41-43, 81-81, and 160-162 showed slowerexchange only when huE104.v2 was bound (Table 10). The most intriguingdifferences in slower hydrogen exchange are the amide bonds of residues81-83 (081, L82, and L83 in chymotrypsinogen numbering, corresponding toQ100, L101, and L102 of SEQ ID NO: 71) in tryptase, which only showedslower exchange when huE104.v2 is bound. This area is particularlyinteresting since it is part of the loop in which two critical contactresidues (Y74 and Y75) of the small interface reside. Tryptase residues81-83 are also in direct contact with HVR-H2 of huE104.v1, as seen inthe crystal structure complex with tetrameric tryptase, but according tothe HDX results it is considered that this area must make a stronger andpresumably different interaction with huE104.v2 than for the interactionof huE104.v1 with tryptase. Conformational changes to the 80s loopCa-backbone and side chains could affect the conformation of Y74 andY75, which are key to the hydrophobic small interface between twotryptase protomers in the tetramer complex. Since the interface issymmetric, residues Y74 and Y75 from both interacting protomers areaffected when huE104.v2 is bound to each protomer, which would amplifyany changes and instability of this interface.

As mentioned above, the differences between huE104.v1 and huE104.v2 arethe vernier framework residues V71 R and F78V. It has been previouslyreported that framework residue changes in position 71 in the heavychain of antibodies affect the conformation of HVR-H2. Modelingexperiments of HVR-H2 in huE104.v1 and huE104.v2 confirm this to be thecase (data not shown). Without being bound by theory, changes in bothvernier residues could affect the conformation of HVR-H2 such that theinteraction with tryptase residues 81 through 83 differs and translatesinto changes of the small interface of the tetramer. Additionally,tryptase amide bonds of residues R69 and E70 (chymotrypsinogennumbering), which are in close structurally proximity, also show slowerHDX only when huE104.v2 is bound. This finding would make changes to the70s loop, which constitutes a significant part of the small interface,even more likely.

When comparing the regions of slower HDX due to Fab binding withtryptase residues within 4A of the Fab E104.v1 as seen in the crystalstructure, a high degree of overlap was observed between both methods,identifying binding epitope residues in the 20s, 40s, 60s, the 80s and100-loop (Table 10). Other areas identified by HDX, such as positions inthe 110s, 160s, 179, 230s and the 245 region of the primary tryptasesequence, are not in direct contact with the antibody according to thecrystal structure, but show a reduction in conformational dynamics inthe presence of huE104.v1 and huE104.v2.

TABLE 10 Amide bonds of tryptase residues that showed slower HDX whenbound to huE104.v1 or huE104.v2 Tryptase Residues Affected(chymotrypsinogen numbering) Contact residues in huE104.v1 huE104.v2crystal structure 26-27 25-27 25-29 — — 38 — 41-43 40-41 47-50 — 5054-55 — — — — — 60-61 60-61 60-62 66-68 66-70 65 — 81-83 81-87 85-88 88— 108-110 108-110 107-110 119-122 — — — 160-162 — 179 — — 230-231 — — —— — 244-245 — —

To summarize these data, huE104.v1 and huE104.v2 have the same HVRsequences and the same contact residues on human tryptase beta 1. Basedon the HDX studies, however, subtle differences in the way the twoantibodies bind to the target were detected. The V71 and F78 in theheavy chain FR3 region of huE104.v1 are shown in FIG. 12B. As indicatedin the figure, the V71R and F78V in the FR3 region of the heavy chain inhuE104.v2 may have affected the position of HVR-H2 and the binding ofHVR-H2 to tryptase, evidenced by the slower HDX in the Q81, L82, and L83region (chymotrypsinogen numbering) when huE104.v2 is bound. The Q81,L82 and L83 residues showed slower HDX when huE104.v2 is bound totryptase as compared to unbound tryptase. As a result, the hydrophobicinteraction of Y75 with the neighboring protomer is weakened. huE104.v1has V at position 71 and F at position 78 (as in the VH_(IV) graft). Inthis context, the HVR-H2 is presented slightly differently, and theeffect of binding of E104.v1 to tryptase, though at the same contactresidues, differs slightly in the dissociation of the small interface,as compared to E104.v2.

In summary, hu31A.v11 Fab and IgG both dissociate tryptase tetramer.Both huE104.v2 Fab and IgG also dissociate tryptase tetramer. huE104.v1IgG, but not Fab, is capable of dissociating tryptase tetramer. However,huE104.v1 IgG at high antibody to tetramer ratio loses inhibitoryactivity. More than one hu31A.v11 Fab binding to the same tetramer wouldcause steric clash, while huE104.v2 Fabs binding to the same tetramer donot. Thus, the hinge region of IgG1 or IgG4 may play a role in orientingthe two Fabs of the huE104.v1 IgG and creates sufficient tension thatresults in the dissociation of the tetramer (data not shown). At highantibody to tetramer ratio, however, huE104.v1 IgG more likely binds totetramer as a monovalent binder, i.e., a Fab, and loses inhibitoryactivity.

C. hu31A.v11 and huE104.v2 Compete for Binding to Human Tryptase Beta 1

The epitope of hu31A.v11 determined by X-ray crystallographysubstantially overlaps with the epitope of huE104.v1 and huE104.v2. Wenext determined whether hu31A.v11 and huE104.v2 compete for binding tohuman tryptase beta 1 by epitope binning. Epitope binning was done usingOCTET® RED384 System (ForteBio, Inc., Menlo Park, Calif., USA). Briefly,human tryptase beta 1 monomer protein was biotinylated at Lys residue byreacting with NHS-PEG4-biotin. Biotinylated monomer was diluted to 5μg/ml in kinetics buffer (ForteBio, Inc., Menlo Park, Calif., USA) andimmobilized onto streptavidin sensor tips (ForteBio, Inc., Menlo Park,Calif., USA). After the immobilization step, human tryptase beta1-immobilized sensors were saturated with the first antibody, diluted at10-20 μg/ml, followed by binding with second antibody diluted at 2.5μg/ml. A binding signal by second antibody implies that the twoantibodies can bind antigen simultaneously at distinct, non-overlappingepitopes, whereas no binding signal implies that they share a commonepitope. Forte Bio data analysis software 8.1 was used to generateepitope-binning matrix.

The results shown in Table 11 below demonstrate that no additionalbinding signal was detected either using huE104.v2 as the first antibodyand hu31A.v11 as the second antibody or vice versa. Either antibodyadded after buffer resulted in additional binding. Thus, the twoantibodies compete for binding to human tryptase beta 1.

TABLE 11 hu31A.v11 and huE104.v2 compete for binding to human betatryptase 1 2nd Antibody 1st Antibody hu31A.v11 IgG4 huE104.v2 IgG4huE104.v2 IgG4 −0.0086 −0.0083 Hu31A.v11 IgG4 −0.0205 0.011 Buffer 0.4230.514

Example 4: Pharmacokinetic (PK) Analysis of Humanized Anti-TryptaseAntibodies

To assess the pharmacodynamics (PK) characteristics of humanizedanti-tryptase antibodies, huE104.v2 or hu31A.v11 IgG4 antibodies wereadministered by intravenous (IV) injection at 1 or 10 mg/kg to C57BL/6mice, n=3. The concentration of anti-gD IgG4, anti-tryptase hu31A.v11IgG4, or anti-tryptase huE104.v2 IgG4 in C57-BL6 mouse serum wasdetermined with a generic immunoglobulin pharmacokinetic ELISA usingsheep-anti-human-IgG (The Binding Site; San Diego, Calif.) for captureand HRP-conjugated-sheep anti-human-IgG (Bethyl Laboratories,Montgomery, Tex.), for detection. The assay sensitivity was 15.6 ng/mLin serum. The huE104.v2 and hu31A.v11 IgG4 antibodies exhibited similarPK, characterized by dose-proportional and linear PK across the doserange tested (FIG. 13 and Table 12). In addition, both antibodies had arelatively low clearance (˜5 ml/day/kg), suggesting that both antibodiesbehave well after single dose administration and are well within theaccepted range. In other experiments, huE104.v2 IgG1 performed similarlyto the huE104.v2 IgG4 and hu31A.v11 IgG4 antibodies (data not shown).

TABLE 12 PK analysis of humanized anti-tryptase antibodies in miceAnti-Tryptase AUC_(last) Clearance C_(max) IgG4 Dose (day • μg/ml)(ml/day/kg) (μg/ml) huE104.v2  1 mg/kg 127 5.00 21.6 huE104.v2 10 mg/kg1400 5.12 242 hu31A.v11  1 mg/kg 155 4.74 25.8 hu31A.v11 10 mg/kg 17504.54 251

The PK characteristics of humanized anti-tryptase antibodies were alsoassessed in male cynomolgus monkeys (cyno), n=3. A control antibody(anti-gD), huE104.v2 IgG4, or hu31A.v11 IgG4 was administered byintravenous (IV) injection at 30 mg/kg (FIG. 14 and Table 13). Theconcentration of anti-gD IgG4, anti-tryptase hu31A.v11 IgG4, oranti-tryptase huE104.v2 IgG4 in cynomolgus monkey serum was determinedwith a Gyrolab XP immunoassay consisting of biotin-conjugated goatanti-human IgG (Bethyl Laboratories, Montgomery, Tex.) capture andAlexa® Fluor 647-conjugated mouse anti-human Fc (R10Z8E9) detection. Theassay sensitivity was 41 ng/mL in serum. Table 13 shows the area underthe curve (AUC), clearance (CL), C_(max), and half-life (T_(1/2)).hu31A.v11 exhibited similar pharmacokinetics to the control anti-gDantibody. A low clearance (CL) of approximately 3 mL/day/kg and T_(1/2)of about 15 days were well within the acceptable range.

TABLE 13 PK analysis of humanized anti-tryptase antibodies in cyno (n =3) AUC CL C_(max) T_(1/2) Group (day • μg/mL) (mL/day/kg) (μg/mL) (days)Anti-gD 11100 ± 1220 2.73 ± 0.285 972 ± 68.8 14.1 ± 1.37 hu31A.v11 10700± 3530 2.99 ± 0.855 805 ± 72.3 14.9 ± 5.95 huE104.v2  7910 ± 2270 3.98 ±1.00  808 ± 48.9 11.5 ± 4.43

Example 5: Formulation of Anti-Tryptase Antibody hu31A.v11 to AmeliorateHVR-H3 Trp100 (W100) Oxidation

While evaluating both hu31A.v11 and huE104.v2 antibodies, it wasunexpectedly discovered that the VH Trp100 residue present in the HVR-H3region of hu31A.v11 (FIG. 1) is susceptible to oxidation, for example,following exposure to 2,2-azobis(2-amidinopropane) dihydrochloride(AAPH) (also referred to as “AAPH stress”) or ambient light (“ambientlight stress”). This oxidation can be considered undesirable in thecontext of a therapeutic antibody. However, the Trp100 residue was foundto be important for binding of hu31A.v1 to tryptase, as well as forinhibitory activity. For instance, as described below, mutation ofTrp100 to mitigate oxidation resulted in variants with reduced bindingaffinity and inhibitory activity. Therefore, oxidation of hu31A.v1,particularly at HVR-H3 W100, was mitigated using formulations containingan antioxidant excipient, e.g., N-acetyltryptophan and/or methionine.

The effect of AAPH stress and hu31A.v1 HVR-H3 W100 (i.e., the tryptophanresidue at position 100 of the VH domain, see FIG. 1) oxidation ontryptase binding and inhibitory activity was evaluated. Samples wereformulated in 1 mM AAPH for 16 h at 40° C. Under these conditions, therewas a 75% increase in W100 oxidation (percent oxidation). The stressedantibody was digested with trypsin and the digested peptides weresubjected to UHPLC-HRMS (ultra high performance liquidchromatography-high resolution mass spectrometry) to determinepercentage of tryptophan oxidation. Briefly, a 250 μg sample ofhu31A.v11 was reduced with 20 mM DTT in 6 M guanidine hydrochloride, 360mM Tris, and 2 mM EDTA at pH 8.6 for 1 hr. The reduced sample was cooledto room temperature and alkylated using 1 M iodoacetic acid (finalconcentration, 50 mM) for 15 min in the dark. The sample was thenbuffer-exchanged into digestion buffer (25 mM Tris, 2 mM CaCl₂), pH8.2). The buffer-exchanged sample was digested with trypsin for 4 hr at37° C. using a 1:40 (w/w) enzyme to antibody ratio. The digestion wasstopped by addition of 100% formic acid to a final concentration of3.0%.

10 μg of the tryptic peptides was injection to a Waters 2.1×150 mm,Acquity UPLC® CSH C18 column with 1.7 μm, 130 Å particles, running at aflow rate of 0.2 mL/min at 77° C. coupled to a Q Exactive massspectrometer system with the following gradient: 1% B (0.1% formic acidin acetonitrile) at 0-2 min; 13% B at 7 min; 35% B at 42 min; 85% B44-46 min; 1% B 46.1 min. The Q Exactive was operated in the datadependent mode, collecting a full MS scan from 200-2000 m/z at 35,000resolution, and an automatic gain control (AGC) target of 1×10⁶. The 8most abundant ions per scan were selected for tandem MS at 17,500resolution and AGC target of 1×10⁵. Relative quantitation of W100oxidation was generated as follows: (1) Peak areas were calculated byintegrating extracted ion chromatograms of the top three most abundantisotopes from charge states with a relative abundance of 10% and abovefor the native tryptic peptide and its oxidized counterparts. (2) Thetotal oxidized peak area was divided by the sum of the oxidized andnative peak areas and multiplied by 100 to obtain the percent oxidation.

The results in Table 14 show that AAPH stress reduced binding ofhu31A.v1 to tryptase monomer as measured by BIAcore® SPR analysis (Table14). Reduced binding was also observed for binding to tryptase tetramer.In contrast, the binding of huE104.v2 to tryptase monomer and tetramerwas not affected by AAPH stress (Table 14). Additionally, AAPH stressreduced the activity of hu31A.v1 in an in vitro tryptase enzymaticactivity assay by about 5-fold, whereas the inhibitory activity ofhuE104.v2 was not affected (data not shown). In summary, after AAPHstress, hu31A.v11 IgG4, which had 75% oxidation at HVR-H3 W100, showedan approximately 6-fold higher K_(D) (i.e., reduced affinity) (with 35%Rmax), and a 5-fold increase in IC50 (i.e., decrease in potency). Rmaxindicates the maximum response in a BIAcore® SPR experiment, and adecrease in Rmax reflects the fact that less of the AAPH-stressedantibody bound to tryptase. In contrast, AAPH stress had minimal, ifany, effect on the binding and potency of huE104.v2 IgG4.

TABLE 14 AAPH stress reduces binding of hu31A.v11 to tryptase TryptaseK_(on) K_(off) K_(D) Sample Beta 1 (1/Ms) (1/s) (M) hu31A.v11 ControlMonomer 5.976E5 1.328E-4 2.222E-10 hu31A.v11 AAPH Monomer 6.076E57.978E-4 1.313E-9  huE104.v2 Control Monomer 5.846E5 1.861E-4 3.183E-10huE104.v2 AAPH Monomer 6.795E5 1.535E-4 2.260E-10

In one approach to reduce HVR-H3 oxidation, variant antibodies havingsubstitutions at HVR-H3 W100 were produced, and the binding of thesevariants to tryptase beta 1 monomers was assessed (Table 15). ResidueW100 of hu31A.v11 was mutated to phenylalanine (F), tyrosine (Y), valine(V), leucine (L), or arginine (R). Surprisingly, all of the variantsshowed reduced binding to tryptase beta 1 monomer. The hu31A.v11 W100Fvariant had the highest affinity for tryptase beta 1 monomer as comparedto the other variants, but exhibited an approximately 100-fold fasteroff-rate (K_(off)) compared to wild-type hu31A.v11, with a similaron-rate (K_(on)) (Table 15). Further, these variants were inferior tohu31A.v11 WT in terms of inhibitory activity (Table 15). For some of thevariants, the inhibitory activity was essentially lost (e.g., for W100R,W100L, and W100V). The hu31A.v11 W100F and W100Y variants inhibitedhuman tryptase beta 1, but had IC50 values that were approximately 2- to3-fold increased (i.e., reduced activity) compared to hu31A.v11 WT. InTable 15, “nd” indicates not detectable or above 1 μM.

TABLE 15 Effect of hu31A.v11 W100 variants on binding affinity totryptase beta 1 monomer and IC50 as compared to hu31A.v11 wild type (asfold increase) hu31A.v11 hu31A.v11 hu31A.v11 hu31A.v11 huE104.v2huE104.v2 (W100) W100F W100Y W100V W100L W100R K_(D) 1 80 234 821 241154 IC50 1 2.5 3.46 nd nd nd

As described above, structural studies revealed that HVR-H3 W100 mademultiple interactions with tryptase, including interaction with R65 oftryptase (chymotrypsinogen numbering), which was determined to be partof the contact residues of hu31A.v11 (see FIG. 8). The Trp at position100 is considered to interact with tryptase to a greater extent than thePhe in the W100F mutant, resulting in higher binding affinity and morepotent inhibitory activity.

In view of the reduced affinity and inhibitory activity of hu31A.v11having oxidation at HVR-H3 W100, as well as the importance of W100 intryptase inhibition, as shown above, an alternative strategy to mitigateoxidation at W100 was pursued. The ability of antioxidant excipients toreduce W100 oxidation was determined. In one example, samples weresubjected to 5 mM AAPH for 24 h at 40° C., with or without exemplaryanti-oxidant excipients, i.e., 0.3 mM N-acetyltryptophan (NAT) and 5 mMmethionine (Met) in formulation containing 0.02% polysorbate 20, 200 mMarginine succinate (pH 5.5), and 150 mg/mL of hu31A.v11 antibody. Theoxidation level of CDR H3 W100 and the potency of the antibody weremeasured based on the chromogenic S-2288 enzymatic assay. The data inTable 16 below show that in this experiment, AAPH stress resulted in 38%oxidation at W100 of hu31A.v11 IgG4, which led to potency loss by 32% ascompared to control, non-stressed antibody sample (n=2). Antibodycomposition in the formulation containing 0.3 mM NAT and 5 mM Met showedreduced oxidation level from 38% to 26%, and reduced potency loss from32% to 21% (n=2). Thus, formulation containing anti-oxidant excipientssuch as NAT and Met reduced AAPH-induced oxidation at W100 and restoredantibody potency. In a separate example, the sample is subjected toambient light stress conditions (60 h at 5000 Lux/h, which is a milderstress condition than AAPH) in the same formulation with or without theanti-oxidant excipients. The mild ambient light stress conditionresulted in 6% oxidation at W100, and the presence of the excipientsfurther reduced the level of oxidation (Table 16). The oxidation levelinduced by the ambient light-induced stress process did not affectantibody potency.

TABLE 16 Effect of antioxidant formulation on hu31A.v11 oxidation Meanrelative potency, HVR-H3 Sample (potency loss) W100 Description (n = 2)oxidation 5 mM AAPH sample,  68% (32%) 38% w/o NAT/MET 5 mM AAPH sample, 79% (21%) 26% 0.3 mM NAT/5 mM MET Light stressed sample, 100% (0%)  6%w/o NAT/MET

In summary, these results show that hu31A.v11 HVR-H3 W100 isunexpectedly susceptible to oxidation. Mutagenesis data showed that W100is important for binding affinity and inhibitory activity of thehu31A.v11 antibody, and none of the tested variants at this residue hadcomparable affinity or inhibitory activity to wild-type hu31A.v11.Antioxidants such as NAT and Met can be used to mitigate the unexpectedoxidation observed at hu31A.v11 HVR-H3 W100, for example, inpharmaceutical antibody formulations for treatment of disorders (e.g.,asthma).

Example 6: Anti-Tryptase Antibody hu31A.v11 Inhibits Tryptase ActivityIn Vivo

A. Materials and Methods

(i) Cyno Active Tryptase ELISA Assay

The concentration of cynomolgus monkey (cyno) active tryptase (tetramer)in biological sample, e.g., bronchoalveolar lavage fluid (BAL), wasdetermined by an ELISA assay. A monoclonal antibody recognizing cynotryptase D1 was utilized as the capture antibody. Recombinant cynoactive tryptase D1 was used as the source material for preparation ofassay standards. Assay standards, controls, and diluted samples wereincubated with 500 μg/ml soybean trypsin inhibitor (SBTI; Sigma Cat. No.10109886001) for 10 min and then labeled with the activity-based probe(ABP; G0353816) for 1 h. See Pan et al. Bioorg. Med. Chem. Lett.16:2882-85, 2006. SBTI was used to bind to the active site in themonomer, which reduces any background caused by active monomer, aspecies that can form in specific in vitro conditions. The SBTI isunable to bind to the active site when the tryptase is in tetramericconformation. A small molecule tryptase inhibitor (G02849855) was addedfor 20 min to stop ABP labeling. A tetramer-dissociating antibody (e.g.,hu31A.v11 IgG4) was added for 10 min to dissociate both ABP-labeled andunlabeled tetramer. This mixture was added to the ELISA plate with acapture antibody for 1 h, washed with 1×PBST, and incubated with theSA-HRP reagent (streptavidin-conjugated horseradish peroxidase, GeneralElectric (GE) catalog number RPN4401V) for 2 h. A colorimetric signalwas generated by applying HRP substrate, tetramethylbenzidine (TMB), andthe reaction was stopped by adding phosphoric acid. The plates were readon a SpectraMax® M5 (Molecular Devices; Sunnyvale, Calif.) plate readerusing 450 nm for detection absorbance and 650 nm for referenceabsorbance. The assay had a reportable range of 20-0.04 ng/mL (in-well),and the assay minimum quantifiable concentration (MQC) was determined tobe 0.08 ng/mL in 1:2 dilution of cyno BAL. Each individual cyno samplewas screened at a single dilution in duplicate. Samples assayed atminimum dilution that fell below the MQC were reported as less thanreportable (LTR).

(ii) Cyno Total Tryptase ELISA Assay

The concentration of cynomolgus monkey (cyno) total tryptase in abiological sample, e.g., BAL, was determined by an ELISA assay. Anantibody recognizing cyno tryptase D1 was utilized as the captureantibody. An antibody recognizing cyno tryptase D1 without competingwith a tetramer-dissociating antibody for binding to cyno tryptase D1was utilized as the detection antibody. Recombinant cyno active tryptaseD1 was used as the source material for preparation of assay standards. Atetramer-dissociating antibody (e.g., hu31A.v11, IgG4) was added for 10min to assay standards, controls, and diluted samples in order todissociate any tetramer present. This mixture was added to the ELISAplate with capture antibody for 2 h and then washed with 1×PBST. Thebiotinylated detection antibody was added for 1 h. Next, SA-HRP reagentwas added for 1 h. A colorimetric signal was generated by TMB, and thereaction was stopped by adding phosphoric acid. The plates were read ona SpectraMax® M5 plate reader using 450 nm for detection absorbance and650 nm for reference absorbance. The assay had a reportable range of20-0.02 ng/mL (in-well), and the assay MQC was determined to be 0.08ng/mL in 1:2 dilution of cyno BAL. Each individual cyno sample wasscreened at a single dilution in duplicate. Samples assayed at minimumdilution that fell below the MQC were reported as less than reportable(LTR).

(iii) Human Active Tryptase ELISA Assay

The concentration of human active tryptase (tetramer) was determined byan ELISA assay. The mouse monoclonal antibody clone B12 recognizinghuman tryptase and capable of dissociating the tryptase tetramer wasutilized as the capture antibody (Fukuoka et al. supra). Otherantibodies that bind human tryptase can also be used. Recombinant humanactive tryptase beta 1 was purified and used as the source material forpreparation of assay standards. Assay standards, controls, and dilutedsamples were incubated with 500 μg/ml SBTI for 10 min and then labeledwith the ABP (G0353816) for 1 h. A small molecule tryptase inhibitor(G02849855) was added for 20 min to stop ABP labeling. This mixture wasadded to the ELISA plate with capture antibody for 1 h, washed with1×PBST, and incubated with SA-HRP reagent for 2 h. A colorimetric signalwas generated by applying TMB, and the reaction was stopped by addingphosphoric acid. The plates were read on a SpectraMax® M5 plate readerusing 450 nm for detection absorbance and 650 nm for referenceabsorbance.

(iv) Human Total Tryptase ELISA Assay

The concentration of human total tryptase was determined by an ELISAassay. An antibody (clone B12) recognizing human tryptase and capable ofdissociating the tryptase tetramer was utilized as the capture antibody.A monoclonal antibody recognizing human tryptase was utilized as thedetection antibody. Recombinant human active tryptase beta 1 waspurified and used as the source material for preparation of assaystandards. Samples were added to the ELISA plate with capture antibodyfor 2 h and then washed with 1×PBST. The biotinylated detection antibodywas added for 1 h. Next, SA-HRP reagent was added for 1 h. Acolorimetric signal was generated by applying TMB, and the reaction wasstopped by adding phosphoric acid. The plates were read on a SpectraMax®M5 plate reader using 450 nm for detection absorbance and 650 nm forreference absorbance.

B. Results

To assess whether anti-tryptase antibodies such as hu31A.v11 targettryptase in vivo and inhibit active tryptase activity, an assay wasdeveloped to measure the amount of active tryptase present in samplessuch as bronchoalveolar lavage fluid (BAL) (e.g., from cyno) or taken bya less invasive method such as nasosorption. An activity-based probe,which includes a label (e.g., biotin), a linker, and a reactive groupthat reacts with active tryptase was developed. This probe selectivelyand covalently binds to active tryptase. See Pan et al. supra. This toolcan be used to measure the amount of active tryptase in a sample (FIG.15). The BAL collection procedure involves instillation of buffer intothe airway and the subsequent recovery of that fluid (which can bevariable) and therefore a normalization factor is needed to comparesamples across timepoints and animals. Because urea is a small moleculewith passive diffusion between vascular and airway compartments, theratio of urea in BAL/urea in serum can be used to normalize across timepoints and across animals. See Pinheiro de Oliveira et al. 2010,Critical Care 14:R39.

To determine whether administration of hu31A.v11 targeted tryptase invivo, a 30 mg/kg dose was administered by intravenous injection tohealthy, unchallenged cyno monkeys, and the amount of active tryptase inBAL was determined. At baseline, active tryptase levels were relativelylow and variable across animals. A trend of decreased levels of activetryptase was observed in BAL after administration of hu31A.v11 inanimals with detectable active tryptase at baseline (FIG. 16).

The effect of hu31A.v11 administration was also assessed in an allergenchallenge model in which cyno monkeys are sensitized to the parasiticnematode worm Ascaris by repeated administration by a variety of routes(FIG. 17). The sensitization phase included administration of Ascarisintraperitoneally and intramuscularly on days 0, 7, 15, 71, 78, and 85;by inhalation on days 29, 50, 120, 184, and 198-205; and intramuscularlyon day 34 (FIG. 17). Wheal flare was assessed on days −7, 57, and 140following intradermal administration on those days. Each animal receivedan optimal dose of Ascaris as determined by ORD (Optimal Response Dose),which was performed to characterize the appropriate dose levels ofAscaris to elicit a desired response in each animal (performed duringthe sensitization phase). Doses included 4, 400, and 4000 μg/ml. Theexperimental phase included a vehicle phase, in which vehicle wasadministered on day 1, followed by administration of Ascaris byinhalation on day 2, followed by sampling of BAL and nasosorption 30 minlater to assess the amount of total and active tryptase (FIG. 17). Fourweeks later, the drug phase included administration of hu31A.v11 on day1, followed by administration of Ascaris on day 2, and sampling of BALand nasosorption 30 min later to assess the amount of total and activetryptase (FIG. 17).

In the Ascaris sensitization model, administration of the anti-tryptaseantibody hu31A.v11 led to a significant decrease in active tryptase inBAL in 5 animals that had detectable active tryptase levels at baseline(FIG. 18A), indicating that this antibody inhibits tryptase activity invivo. Additionally, administration of the anti-tryptase antibody alsoled to an increase in the amount of total tryptase in BAL in all animals(FIG. 18B). Other experiments showed that the level of total tryptase innasal mucosal lining fluid (MLF) was increased after dosing theanti-tryptase antibody hu31A.v11, as assessed by the level of totaltryptase in the nasosorption sample (FIG. 18C). In this experiment, theMLF was collected using a synthetic absorptive matrix (SAM; HuntDevelopments (UK), Ltd, West Sussex, England). The increase of totaltryptase after dosing is indicative of target engagement. Activetryptase was undetectable in the nasosorption sample before and afterdosing. The * in FIGS. 18A and 18C indicate levels below detection.

Next, in vivo proof of activity was also tested in human-engraftedIL2Rgnull-3/GM/SF NOD-SCID mice. A humanized mouse model for human mastcell engraftment and human IgE-dependent allergic responses waspreviously developed. Briefly, NSG-SGM3 mice (Jackson Laboratory, stock#013062) were developed from the NOD.Cg-Prkdc^(scid) II2rg^(tm1Wji)/SzJ(NSG) background, and like their NSG parents, NSG-SGM3 mice lack matureT cells, B cells, and functional NK cells, and are deficient in mousecytokine signaling. These mice contain three co-injected transgenes,each driven by a human cytomegalovirus promoter/enhancer sequence.Triple transgenic NSG-SGM3 mice constitutively produce 2-4 ng/ml serumlevels of human SCF, GM-CSF, and IL-3, providing cell proliferation andsurvival signals (see, e.g., Bryce et al. J. Allergy Clin Immunol.138(3):769-779, 2016). The NSG-SGM3 BLT mice develop human mast cellsthat populate peripheral lymphoid tissues, mucosal tissues, and theperitoneal cavity. The human mast cells in NSG-SGM3 BLT mice expressCD117, tryptase, and IgE-receptor, and can undergo a calcium flux anddegranulate in an IgE-dependent antigen-specific manner. Upon challenge,the NSG-SGM3-BLT mice develop a human mast cell dependent,antigen-specific IgE-mediated passive systemic anaphylaxis response thatcan be analyzed by measuring changes in body temperature.

The experiment was designed to examine the ability of anti-tryptaseantibodies in inhibiting IgE-mediated passive systemic anaphylaxis inthe engrafted mice. 10-12 weeks old NSG-SGM3 mice (Jackson Laboratorystock #013062) were divided into three groups: Group 1: NSG-SGM3 micewere treated intraperitoneally (i.p.) with isotype antibody (500μg/mice); Group 2: NSG-SGM3 mice were treated with human anti-tryptaseantibody (hu31A.v11 IgG4, 13.3 mg/mL) i.p. (500 μg/mice); and Group 3:NSG-SGM3 mice were treated with tryptase small molecule inhibitorG02849855 (30 mg/kg) i.p. On day −1, all mice were shaved clean ofabdominal hair to facilitate body temperature measurement. On day 0,animals were injected with either control isotype antibody oranti-tryptase antibody. Dose volume was formulated in 100 μL.Anti-tryptase antibody was dissolved in 200 μL saline for injection. 15min after treatment, mice from Groups 1-3 were sensitized intravenouslywith anti-NP (4-Hydroxy-3-nitrophenylacetyl hapten) IgE JW8.5.13 (SigmaCat. No. 87080706-1VL; see also US20070253948 and Jackman et al. J.Biol. Chem. 285(27):20850-20859, 2010),1.6 μg in 200 μL of saline. Onday 1 (24 h after treatment), body temperature was measured by manuallyrestraining the mice and placing a Braun ThermoScan® Pro 4000 firmlyagainst the shaved abdominal skin just below the sternum. Immediatelyafter baseline body temperature measurement, mice were challenged i.v.with 500 μg of the antigen NP conjugated to a carrier protein BSA(NP-BSA) in 200 μL saline. Every 15 minutes after challenge, bodytemperature will be measured for at least 60 minutes.

As shown in FIG. 19, mice treated with the anti-tryptase antibodyhu31A.v11 showed an improved body temperature maintenance as compared tomice treated with a control anti-gD antibody, upon IgE challenge.

These data show that the anti-tryptase antibody hu31A.v11 is active andcan bind and inhibit tryptase activity in vivo. These data providefurther evidence that anti-tryptase antibodies such as hu31A.v1 can beused as therapeutic agents for treatment of tryptase-associateddisorders such as asthma.

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

IV. Sequence Listing

Table 17 shows sequences that are used throughout the application.

TABLE 17 Sequence Listing SEQ ID NO: Sequence 1X₁X₂GMX₃, wherein X₁ is Asp or Ser, X₂ is Tyr or Phe, and X₃ is Val orHis 2 FISSGSSTVYYADTMKG 3RX₁X₂X₃DWYFDV, wherein X₁ is Asn or Asp, X₂ is Tyr or Asn, and X₃ isAsp or Tyr 4 SASSSVTYMY 5 RTSDLAS 6 QHYHSYPLT 7 DYGMV 8 RNYDDWYFDV 9EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRNYDDWYFDVWGQGTLVTVSS 10DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKSPKPWIYRTSDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYHSYPLTFGQGTKVEIK 11 EVQLVESGGGLVQPGGSLRLSCAASGFTFS12 WVRQAPGKGLEWVA 13 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR 14 WGQGTLVTVSS 15DIQMTQSPSSLSASVGDRVTITC 16 WYQQKPGKSPKPWIY 17GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 18 FGQGTKVEIK 19EVKLVESGGGSVQPGGSRKLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNPKNTLFLQMSSLRSEDTAMYYCARRDNYDWYFDVWGTGTTVTVSS 20QIVLTQSPAIMSASPGEKVTISCSASSSVTYMYWYQQKPGSSPKPWIYRTSDLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQHYHSYPLTFGAGTKLELK 21 EVKLVESGGGSVQPGGSRKLSCAASGFTFS22 WVRQAPGKGLEWVA 23 RFTISRDNPKNTLFLQMSSLRSEDTAMYYCAR 24 WGTGTTVTVSS 25QIVLTQSPAIMSASPGEKVTISC 26 WYQQKPGSSPKPWIY 27GVPARFSGSGSGTSYSLTISSMEAEDAATYYC 28 FGAGTKLELK 29 RDNYDWYFDV 30 GYAIT 31GISSAATTFYSSWAKS 32 DPRGYGAALDRLDL 33 QSIKSVYNNRLG 34 ETSILTS 35AGGFDRSGDTT 36EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSS 37DIQMTQSPSSLSASVGDRVTITCQSIKSVYNNRLGWYQQKPGKAPKLLIYETSILTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGFDRSGDTTFGQGTKVEIK 38EVQLVESGPGLVKPSETLSLTCTVSRFSLI 39WX₁RQPPGKGLEWIG, wherein X₁ is Ile or Val 40RX₁TISX₂DTSKNQX₃SLKLSSVTAADTAVYX₄CAR, wherein X₁ is Val or Ser, X₂ isArg or Val, X₃ is Val or Phe, and X₄ is Tyr or Phe 41 WGQGTLVTVSS 42WIRQPPGKGLEWIG 43 RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR 44EVQLVESGGGLVQPGGSLRLSCAVSRFSLI 45 WVRQAPGKGLEWIG 46RSTISRDTSKNTVYLQMNSLRAEDTAVYFCAR 47EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSS 48EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWVRQPPGKGLEWIGGISSAATTFYSSWAKSRSTISRDTSKNQVSLKLSSVTAADTAVYFCARDPRGYGAALDRLDLWGQGTLVTVSS 49EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISVDTSKNQVSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSS 50EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSS 51EVQLVESGGGLVQPGGSLRLSCAVSRFSLIGYAITWVRQAPGKGLEWIGGISSAATTFYSSWAKSRSTISRDTSKNTVYLQMNSLRAEDTAVYFCARDPRGYGAALDRLDLWGQGTLVTVSS 52QSLEESGGGLFKPTDTLTLTCTVSRFSLIGYAITWVRQSPENGLEWIGGISSAATTFYSSWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCARDPRGYGAALDRLDLWGQGTLVTVSS 53AAVLTQTPASVSAAVGGTVSISCQSIKSVYNNRLGWYQQKPGQPPKLLIYETSILTSGVPSRFKGSGSETQFTLTISDVQCDDAATYFCAGGFDRSGDTTEGGGTEVVVK 54QSLEESGGGLFKPTDTLTLTCTVSRFSLI 55 WVRQSPENGLEWIG 56RSTITRNTNENTVTLKMTSLTAADTATYFCAR 57 WGQGTLVTVSS 58DAQLTQSPSSLSASVGDRVTITCQSIKSVYNNRLGWYQQKPGKPPKLLIYETSILTSGVPSRFSGSGSETDFTLTISSLQPEDFATYFCAGGFDRSGDTTFGQGTKVEIK 59AAVLTQTPASVSAAVGGTVSISCQSIKSVYNNRLGWYQQKPGQPPKLLIYETSILTSGVPSRFKGSGSETQFTLTISDVQADDAATYFCAGGFDRSGDTTEGGGTEVVVK 60DX₁QX₂TQSPSSLSASVGDRVTITC, wherein X₁ is Ile or Ala, and X₂ is Met orLeu 61 WYQQKPGKX₁PKLLIY, wherein X₁ is Ala or Pro 62GVPSRFSGSGSX₁TDFTLTISSLQPEDFATYX₂C, wherein X₁ is Gly or Glu, and X₂is Tyr or Phe 63 FGQGTKVEIK 64 DIQMTQSPSSLSASVGDRVTITC 65WYQQKPGKAPKLLIY 66 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 67AAVLTQTPASVSAAVGGTVSISC 68 WYQQKPGQPPKLLIY 69GVPSRFKGSGSETQFTLTISDVQX₁DDAATYFC, wherein X₁ is Cys or Ala 70FGGGTEVVVK 71MLNLLLLALPVLASRAYAAPAPGQALQRVGIVGGQEAPRSKWPWQVSLRVHGPYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKP 72MLNLLLLALPVLASRAYAAPAPGQALQRVGIVGGQEAPRSKWPWQVSLRVHGPYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVKVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKP 73MLNLLLLALPVLASRAYAAPAPGQALQRVGIVGGQEAPRSKWPWQVSLRVRDRYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQVATAPHTFPAPS 74 GATGGTGACTGTTCCAGTTGC 75CATTGGTGAGGGTGCCCGAGTTC 76EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRNYDDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 77DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKSPKPWIYRTSDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYHSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 78EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRNYDDWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 79DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKSPKPWIYRTSDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYHSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 80EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 81DIQMTQSPSSLSASVGDRVTITCQSIKSVYNNRLGWYQQKPGKAPKLLIYETSILTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGFDRSGDTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 82EVQLVESGPGLVKPSETLSLTCTVSRFSLIGYAITWIRQPPGKGLEWIGGISSAATTFYSSWAKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCARDPRGYGAALDRLDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 83DIQMTQSPSSLSASVGDRVTITCQSIKSVYNNRLGWYQQKPGKAPKLLIYETSILTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGFDRSGDTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 84 AYSVN 85 MIWGDGKIVYNSALKS 86 DGYYPYAMDN 87 RASKSVDSYGNSFMH88 LASNLES 89 QQNNEDPRT 90QVTLRESGPALVKPTQTLTLTCTVSGFSLSAYSVNWIRQPPGKALEWLAMIWGDGKIVYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAGDGYYPYAMDNWGQGSLVTVSS 91DIVMTQSPDSLSVSLGERATINCRASKSVDSYGNSFMHWYQQKPGQPPKLLYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPRTFGGGTKVEIK 92DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPFTFGQGTKVEIK 93EVQLVESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARFDYWGQFTLVTVSS 94EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVGAISSSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFDYWGQGTLVTVSS 95TLTISSLQPEDFATYYCQQYYSYPFTFGQGTKVEIK 96TLTISDVQCDDAATYFCAGGFDRSGDTTFGGGTEVVVK 97IVGGQEAPRSKWPWQVSLRVHGPYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKP 98EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVGFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVVYCARRDNYDWYFDVWGQGTLVTVSS 99EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVVYCARRDNYDWYFDVWGQGTLVTVSS 100EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVVYCARRDNYDWYFDVWGQGTLVTVSS 101EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMVWVRQAPGKGLEWVAFISSGSSTVYYADTMKGRFTISRDNSKNTLYLQMNSLRAEDTAVVYCTRRDNYDWYFDVWGQGTLVTVSS 102DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYRTSDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYHSYPLTFGQGTKVEIK 103DIQLTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKSPKPWIYRTSDLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHYHSYPLTFGQGTKVEIK 104GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCAGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGATTATGGCATGGTGTGGGTGCGCCAGGCCCCAGGCAAAGGCCTGGAATGGGTGGCCTTCATCAGCAGCGGCAGCAGCACCGTGTATTATGCCGATACCATGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTATTATTGCACCCGCCGCAACTACGATGATTGGTATTTCGATGTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGT 105GATATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATCACCTGCAGCGCCAGCAGCAGCGTGACCTATATGTATTGGTATCAGCAGAAACCAGGCAAAAGCCCAAAACCATGGATCTATCGCACCAGCGATCTGGCCAGCGGCGTGCCAAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGATTTCACCCTGACCATCAGCAGCCTGCAGCCAGAAGATTTCGCCACCTATTATTGCCAGCACTATCACAGCTATCCACTGACCTTCGGCCAGGGTACCAAGGTGGAGATCAAA 106GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCAGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGATTATGGCATGGTGTGGGTGCGCCAGGCCCCAGGCAAAGGCCTGGAATGGGTGGCCTTCATCAGCAGCGGCAGCAGCACCGTGTATTATGCCGATACCATGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTATTATTGCACCCGCCGCAACTACGATGATTGGTATTTCGATGTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 107GATATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATCACCTGCAGCGCCAGCAGCAGCGTGACCTATATGTATTGGTATCAGCAGAAACCAGGCAAAAGCCCAAAACCATGGATCTATCGCACCAGCGATCTGGCCAGCGGCGTGCCAAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGATTTCACCCTGACCATCAGCAGCCTGCAGCCAGAAGATTTCGCCACCTATTATTGCCAGCACTATCACAGCTATCCACTGACCTTCGGCCAGGGTACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 108GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCAGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGATTATGGCATGGTGTGGGTGCGCCAGGCCCCAGGCAAAGGCCTGGAATGGGTGGCCTTCATCAGCAGCGGCAGCAGCACCGTGTATTATGCCGATACCATGAAAGGCCGCTTCACCATCAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCCGAAGATACCGCCGTGTATTATTGCACCCGCCGCAACTACGATGATTGGTATTTCGATGTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGCTCCCGCAGTACTTCTGAGTCCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCAAGACCTACACGTGCAACGTGGATCACAAGCCCAGCAACACCAAGGTGGACAAACGCGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 109GAAGTGCAGCTGGTGGAAAGCGGCCCAGGCCTGGTGAAACCAAGCGAAACCCTGAGCCTGACCTGCACCGTGAGCCGCTTCAGCCTGATCGGCTATGCCATCACCTGGATCCGCCAGCCACCAGGCAAAGGCCTGGAATGGATCGGCGGCATCAGCAGCGCCGCCACCACCTTCTATAGCAGCTGGGCCAAAAGCCGCGTGACCATCAGCCGCGATACCAGCAAAAACCAGGTGAGCCTGAAACTGAGCAGCGTGACCGCCGCCGATACCGCCGTGTATTATTGCGCCCGCGATCCACGCGGCTATGGCGCCGCCCTGGATCGCCTGGATCTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGT 110GATATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATCACCTGCCAGAGCATCAAAAGCGTGTATAACAACCGCCTGGGCTGGTATCAGCAGAAACCAGGCAAAGCCCCAAAACTGCTGATCTATGAAACCAGCATCCTGACCAGCGGCGTGCCAAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGATTTCACCCTGACCATCAGCAGCCTGCAGCCAGAAGATTTCGCCACCTATTATTGCGCCGGCGGCTTCGATCGCAGCGGCGATACCACCTTCGGCCAGGGTACCAAGGTGGAGATCAAA 111GAAGTGCAGCTGGTGGAAAGCGGCCCAGGCCTGGTGAAACCAAGCGAAACCCTGAGCCTGACCTGCACCGTGAGCCGCTTCAGCCTGATCGGCTATGCCATCACCTGGATCCGCCAGCCACCAGGCAAAGGCCTGGAATGGATCGGCGGCATCAGCAGCGCCGCCACCACCTTCTATAGCAGCTGGGCCAAAAGCCGCGTGACCATCAGCCGCGATACCAGCAAAAACCAGGTGAGCCTGAAACTGAGCAGCGTGACCGCCGCCGATACCGCCGTGTATTATTGCGCCCGCGATCCACGCGGCTATGGCGCCGCCCTGGATCGCCTGGATCTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 112GATATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATCACCTGCCAGAGCATCAAAAGCGTGTATAACAACCGCCTGGGCTGGTATCAGCAGAAACCAGGCAAAGCCCCAAAACTGCTGATCTATGAAACCAGCATCCTGACCAGCGGCGTGCCAAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGATTTCACCCTGACCATCAGCAGCCTGCAGCCAGAAGATTTCGCCACCTATTATTGCGCCGGCGGCTTCGATCGCAGCGGCGATACCACCTTCGGCCAGGGTACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 113GAAGTGCAGCTGGTGGAAAGCGGCCCAGGCCTGGTGAAACCAAGCGAAACCCTGAGCCTGACCTGCACCGTGAGCCGCTTCAGCCTGATCGGCTATGCCATCACCTGGATCCGCCAGCCACCAGGCAAAGGCCTGGAATGGATCGGCGGCATCAGCAGCGCCGCCACCACCTTCTATAGCAGCTGGGCCAAAAGCCGCGTGACCATCAGCCGCGATACCAGCAAAAACCAGGTGAGCCTGAAACTGAGCAGCGTGACCGCCGCCGATACCGCCGTGTATTATTGCGCCCGCGATCCACGCGGCTATGGCGCCGCCCTGGATCGCCTGGATCTGTGGGGCCAGGGCACCCTGGTGACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGCTCCCGCAGTACTTCTGAGTCCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCAAGACCTACACGTGCAACGTGGATCACAAGCCCAGCAACACCAAGGTGGACAAACGCGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 114EVTLRESGPALVKPTQTLTLTCTVSGFSLSAYSVNWIRQPPGKALEWLAMIWGDGKIVYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAGDGYYPYAMDNWGQGSLVTVSS 115DIVLTQSPDSLSVSLGERATINCRASKSVDSYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPRTFGGGTKVEIK 116IVGGQEAPRSKWPWQVSLRVHGPYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVKVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKPGNSDYKDDDDK 117IVGGQEAPRSKWPWQVSLRVRDRYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKPGNSDYKDDDDK 118IVGGQEAPRSKWPWQVSLRVRDRYWMHFCGGSLIHPQWVLTAAHCLGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYIIQTGADIALLELEEPVNISSRVHTVMLPPASETFPPGMPCWVTGWGDVDNDEPLSPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIIRDDMLCAGNSQRDSCKGDSGGPLVCKVNGTWLQAGVVSWDEGCAQPNRPGIYTRVTYYLDWIHHYVPKKPGNSDYKDDDDK 119ALPVLVSPAHAAPAPGQALQRVGIVGGKEAPRSKWPWQVSLRLHGQYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLADLRVQLREQHLYYQDQLLPVSRIIVHPQFYAVQIGADIALLELEEPVNVSSHVHTVTLPPALETFPPGTPCWVTGWGDVDNDVRLPPPYPLKEVEVPIVENQLCDAEYHTGLHTGDSFRIVRDDMLCAGSEKHDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCALPNRPGIYTRVTYYLDWIHRYVPEKP 120 SFSMS121 TISGGKTFTDYVDSVKG 122 ANYGNWFFEV 123 RASESVAKYGLSLLN 124 AASNRGS 125QQSKEVPFT 126EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVATISGGKTFTDYVDSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCTRANYGNWFFEVWGQGTLVTVSS 127EIVLTQSPATLSLSPGERATLSCRASESVAKYGLSLLNWFQQKPGQPPRLLIFAASNRGSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPFTFGQGTKVEIK 128AGSTHHHHHHDDDDKIVGGQEAPRSKWPWQVSLRVHGPYWMHFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYTAQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFPLKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQGDSGGPLVCKVNGTWLQAGVVSWGEGCAQPNRPGIYTRVTYYLDWIHHYVPKKP

What is claimed is:
 1. A method of treating a pulmonary disorder in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises the following six hypervariable regions(HVRs): (a) an HVR-H1 comprising the amino acid sequence of DYGMV (SEQID NO: 7); (b) an HVR-H2 comprising the amino acid sequence ofFISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).2. The method of claim 1, wherein the pulmonary disorder is asthma,airway hyperresponsiveness, or chronic obstructive pulmonary disease(COPD).
 3. The method of claim 2, wherein the pulmonary disorder isasthma.
 4. The method of claim 1, wherein the method further comprisesadministering an additional therapeutic agent to the subject.
 5. Themethod of claim 4, wherein the additional therapeutic agent is aninterleukin-13 (IL-13) axis binding antagonist, an interleukin-5 (IL-5)axis binding antagonist, an interleukin-33 (IL-33) axis bindingantagonist, an M1 prime antagonist, an IgE antagonist, a TRPA1antagonist, a CRTH2 antagonist, a broncodilator or asthma symptomcontroller medication, an immunomodulator, a corticosteroid, a Th2pathway inhibitor, a tyrosine kinase inhibitor, or a phosphodiesteraseinhibitor.
 6. The method of claim 1, wherein the antibody isadministered subcutaneously, intravenously, intramuscularly, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally.
 7. The method of claim 1, wherein the antibody comprises(a) a heavy chain variable (VH) domain comprising an amino sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 9; (b) a light chain variable (VL) domain comprising an aminoacid sequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 10; or (c) a VH domain as in (a) and a VL domainas in (b).
 8. The method of claim 1, wherein the VH domain comprises theamino acid sequence of SEQ ID NO:
 9. 9. The method of claim 1, whereinthe VL domain comprises the amino acid sequence of SEQ ID NO:
 10. 10.The method of claim 1, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 9 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 10. 11. The method ofclaim 1, wherein the antibody comprises (a) a heavy chain comprising theamino acid sequence of SEQ ID NO: 76 or SEQ ID NO:78 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO:
 77. 12. Themethod of claim 1, wherein the antibody is capable of inhibiting theenzymatic activity of human tryptase beta
 1. 13. The method of claim 12,wherein the antibody binds the tryptase with a K_(D) of between about120 pM and about 0.5 nM.
 14. The method of claim 13, wherein theantibody binds tryptase with a K_(D) of about 400 pM.
 15. The method ofclaim 12, wherein the antibody inhibits the activity of tryptase with anIC50 of about 2.5 nM or lower as determined by a human tryptase betaenzymatic assay using a colorimetric synthetic peptide substrate. 16.The method of claim 12, wherein: (i) the antibody is capable ofinhibiting the enzymatic activity of human tryptase beta 1 at pH 6; (ii)the antibody is capable of inhibiting tryptase-mediated stimulation ofbronchial smooth muscle cell proliferation and/or collagen-basedcontraction; (iii) the antibody is capable of inhibiting mast cellhistamine release; (iv) the antibody is capable of inhibitingIgE-triggered histamine release and/or tryptase-triggered histaminerelease; (v) the antibody is capable of inhibiting tryptase activity incynomolgus monkey bronchoalveolar lavage (BAL) or nasosorption samples;(vi) the antibody is capable of dissociating tetrameric human tryptasebeta 1; (vii) the antibody is capable of dissociating tetrameric humantryptase beta 1 when in a monovalent format; and/or (viii) the antibodyis capable of dissociating tetrameric human tryptase beta 1 in thepresence of heparin.
 17. The method of claim 1, wherein the antibody iscapable of dissociating both the small interface of tetrameric humantryptase beta 1 and the large interface of tetrameric human tryptasebeta
 1. 18. The method of claim 1, wherein the antibody further bindscynomolgus monkey tryptase, human tryptase alpha, human tryptase beta 2and/or human tryptase beta
 3. 19. The method of claim 1, wherein theantibody is humanized.
 20. The method of claim 1, wherein the antibodyis an IgG antibody.
 21. The method of claim 20, wherein the IgG antibodyis an IgG1 antibody or an IgG4 antibody.
 22. The method of claim 21,wherein the IgG4 antibody comprises an S228P mutation in the heavy chainconstant region according to the EU numbering system.
 23. The method ofclaim 1, wherein the antibody is a monospecific antibody.
 24. The methodof claim 1, wherein the antibody is a multispecific antibody.
 25. Themethod of claim 24, wherein the multispecific antibody is a bispecificantibody.
 26. The method of claim 25, wherein the antibody comprises afirst binding domain that binds to human tryptase beta 1 and a secondbinding domain that binds to a second biological molecule, wherein thesecond biological molecule is IL-13, interleukin-4 (IL-4), IL-5,interleukin-17 (IL-17), IgE, or IL-33.
 27. The method of claim 1,wherein the antibody is comprised in a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, excipient, or diluent.28. A method of treating an autoimmune disorder in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of an isolated antibody that binds tohuman tryptase beta 1, or an antigen-binding fragment thereof, whereinthe antibody comprises the following six HVRs: (a) an HVR-H1 comprisingthe amino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2);(c) an HVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ IDNO: 8); (d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY(SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acid sequence ofRTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acidsequence of QHYHSYPLT (SEQ ID NO: 6).
 29. The method of claim 28,wherein the autoimmune disorder is rheumatoid arthritis, psoriasis,eosinophilic esophagitis, inflammatory bowel disease (IBD), or Crohn'sdisease.
 30. A method of treating an inflammatory disorder in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of an isolated antibody that binds tohuman tryptase beta 1, or an antigen-binding fragment thereof, whereinthe antibody comprises the following six HVRs: (a) an HVR-H1 comprisingthe amino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2);(c) an HVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ IDNO: 8); (d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY(SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acid sequence ofRTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acidsequence of QHYHSYPLT (SEQ ID NO: 6).
 31. The method of claim 30,wherein the inflammatory disorder is chronic spontaneous urticaria(CSU), anaphylaxis, anaphylactic shock, atopic dermatitis, or allergicrhinitis.
 32. A method of treating a fibrotic disorder in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of an isolated antibody that binds tohuman tryptase beta 1, or an antigen-binding fragment thereof, whereinthe antibody comprises the following six HVRs: (a) an HVR-H1 comprisingthe amino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2);(c) an HVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ IDNO: 8); (d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY(SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acid sequence ofRTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acidsequence of QHYHSYPLT (SEQ ID NO: 6).
 33. The method of claim 32,wherein the fibrotic disorder is idiopathic pulmonary fibrosis.
 34. Amethod of treating a granulocytic (neutrophilic or eosinophilic)disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anisolated antibody that binds to human tryptase beta 1, or anantigen-binding fragment thereof, wherein the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprising the amino acid sequenceof FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).35. The method of claim 34, wherein the granulocytic (neutrophilic oreosinophilic) disorder is COPD, asthma-COPD overlap syndrome (ACOS), orCOPD-obstructive sleep apnea (OSA) overlap syndrome.
 36. A method oftreating a monocytic disorder in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of an isolated antibody that binds to human tryptase beta 1, oran antigen-binding fragment thereof, wherein the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprising the amino acid sequenceof FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).37. The method of claim 36, wherein the monocytic disorder is arthritis,esophagitis, allergic rhinitis, non-allergic rhinitis, rhinosinusitiswith polyps, nasal polyposis, bronchitis, chronic pneumonia, allergicbronchopulmonary aspergillosis, airway inflammation, allergic rhinitis,bronchiectasis, chronic bronchitis, a gastrointestinal inflammatorycondition, lupus, or Systemic Lupus Erythematosus (SLE).
 38. A method oftreating a lymphocytic disorder in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of an isolated antibody that binds to human tryptase beta 1, oran antigen-binding fragment thereof, wherein the antibody comprises thefollowing six hypervariable regions (HVRs): (a) an HVR-H1 comprising theamino acid sequence of DYGMV (SEQ ID NO: 7); (b) an HVR-H2 comprisingthe amino acid sequence of FISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) anHVR-H3 comprising the amino acid sequence of RNYDDWYFDV (SEQ ID NO: 8);(d) an HVR-L1 comprising the amino acid sequence of SASSSVTYMY (SEQ IDNO: 4); (e) an HVR-L2 comprising the amino acid sequence of RTSDLAS (SEQID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence ofQHYHSYPLT (SEQ ID NO: 6).
 39. The method of claim 38, wherein thelymphocytic disorder is arthritis, esophagitis, allergic rhinitis,non-allergic rhinitis, rhinosinusitis with polyps, nasal polyposis,bronchitis, chronic pneumonia, allergic bronchopulmonary aspergillosis,airway inflammation, allergic rhinitis, bronchiectasis, chronicbronchitis, a gastrointestinal inflammatory condition, lupus, or SLE.40. A method of treating a disorder associated with increased numbers ordistribution of normal or aberrant tissue resident cells or stromalcells in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of an isolatedantibody that binds to human tryptase beta 1, or an antigen-bindingfragment thereof, wherein the antibody comprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of DYGMV (SEQ ID NO:7); (b) an HVR-H2 comprising the amino acid sequence ofFISSGSSTVYYADTMKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of RNYDDWYFDV (SEQ ID NO: 8); (d) an HVR-L1 comprising theamino acid sequence of SASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2comprising the amino acid sequence of RTSDLAS (SEQ ID NO: 5); and (f) anHVR-L3 comprising the amino acid sequence of QHYHSYPLT (SEQ ID NO: 6).41. The method of claim 40, wherein the disorder is associated withincreased numbers or distribution of mast cells, macrophages,lymphocytes, fibroblasts, myofibroblasts, smooth muscle cells,epithelia, or endothelia.
 42. A method of treating mastocytosis in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of an isolated antibody thatbinds to human tryptase beta 1, or an antigen-binding fragment thereof,wherein the antibody comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of DYGMV (SEQ ID NO: 7); (b) anHVR-H2 comprising the amino acid sequence of FISSGSSTVYYADTMKG (SEQ IDNO: 2); (c) an HVR-H3 comprising the amino acid sequence of RNYDDWYFDV(SEQ ID NO: 8); (d) an HVR-L1 comprising the amino acid sequence ofSASSSVTYMY (SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acidsequence of RTSDLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising theamino acid sequence of QHYHSYPLT (SEQ ID NO: 6).
 43. The method of claim42, wherein the mastocytosis is nonacute systemic mastocytosis.